Method for treatment of insulin resistance in obesity and diabetes

ABSTRACT

Disclosed is a method to identify compounds useful for reducing insulin resistance in a patient, and particularly a patient that has insulin resistance associated with obesity and/or type II diabetes. Also disclosed is a method of reducing insulin resistance in a patient by administering a compound identified using the method of the invention, and particularly, by administering an antagonist of melanocortin stimulating hormone (MSH) biological activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority under 35 U.S.C. § 119(e) from U.S.Provisional Application Serial No. 60/232,292, filed Sep. 13, 2000,entitled, “Method for Investigating and Treating Diabetes”. The entiredisclosure of U.S. Provisional Application Serial No. 60/232,292 isincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a non-human animal model forobesity and uses of such an animal for studying and developing methodsfor identifying compounds for use in the regulation of insulinresistance in obesity and type II diabetes, as well as a method oftreating insulin resistance in obesity and type II diabetes byadministration of such compounds.

BACKGROUND OF THE INVENTION

[0003] Diabetes, and conditions related thereto, are major healthconcerns throughout the world, and, particularly in the United States,contribute to morbidity and mortality. Non-insulin dependent diabetesmellitus (NIDDM), also known as type II diabetes, is the major form ofdiabetes in developed countries. While a large number of environmentaland genetic factors contribute to the risk of NIDDM in the UnitedStates, prolonged obesity is by far the largest risk factor. Themolecular basis of this association, however, is not fully understood.As a consequence, efficient means of therapeutical intervention arelacking.

[0004] Before the development of diabetes, many obese patients develop aperipheral resistance to the actions of insulin. The molecular basis ofinsulin-resistance in obesity has been the subject of intensive study,by nonetheless remains elusive. Insights into components and mechanismsof the link between obesity and insulin resistance have been gained frommouse models of obesity which display obesity-induced insulinresistance. The molecular basis of the various mouse obesity modelscovers a range of mechanisms; nonetheless these all develop diabetes,either before or after the onset of obesity.

[0005] Obesity in humans and rodents is commonly associated with insulinresistance, (i.e., smaller than expected responses to a given dose ofinsulin) (LeRoith et al., Diabetes Mellitus: a Fundamental and ClinicalText. (Lippincott-Raven, Philadelphia, 1996); DeFronzo et al., DiabetesCare 15:318-68 (1992); Rifkin et al., Diabetes Mellitus, (Elsevier,N.Y., 1990)). The mechanisms linking obesity and insulin resistance arenot known. Studies on the potential mechanistic basis of obesity-inducedinsulin resistance have revealed numerous potential sites, making asingle basic mechanism for explaining insulin insensitivity unlikely(Rifkin et al., Diabetes Mellitus, (Elsevier, N.Y., 1990)). Both insulinsecretion and action can be impaired. Accordingly, sites at theanatomical, cellular, and molecular level are the β-cells of thepancreas, and membrane carriers and enzymes regulating metabolicpathways in liver, fat, and muscle. An example for impaired insulinsecretion can be found in a rodent model of obesity withnon-insulin-dependent diabetes mellitus, the Zucker diabetic fatty(fa/fa) rat, where overaccumulation of triglycerides in the pancreaticislets leads to gradual depletion of β cells (Lee et al., Proc Nail AcadSci USA 91:10878-82 (1994); Shimabukuro et al., Proc Natl Acad Sci USA95:2498-502 (1998)). Insulin action can be impaired in a number of ways,involving insulin sensitive carriers or pathways, or the insulinreceptor directly. Earlier studies indicated that quantitativeregulation of the insulin sensitive glucose transporters (Glut-4) maycontribute to insulin resistance; however, this factor alone is probablyinadequate to explain the extent of insulin resistance. For instance,mutant mice lacking Glut-4 develop only mild hyperinsulinemia (Katz etal., Nature 377:151-5 (1995)). More recently studies have focused ondefects at the level of the insulin receptors themselves and atpost-receptor events in type 2 diabetes, specifically the intrinsiccatalytic activity of the insulin receptor and downstream signalingevents. A reduction in tyrosine phosphorylation of both the insulinreceptor (IR) and the insulin receptor substrate-1 (IRS-1) has beennoted in both animals and humans with type 2 diabetes (LeMarchand-Brustel et al., J Recept Signal Transduct Res 19:217-28(1999)). Importantly, this occurs in all of the major insulin-sensitivetissues, namely the muscle, fat and liver. Disruption of IRS-2 in miceimpairs both peripheral insulin signaling and pancreatic β-cell function(Withers et al., Nature 391:900-4 (1998)). Activation ofphosphatidylinositol 3-kinase (PI 3-kinase) was found to be profoundlyaffected in response to insulin (Kerouz et al., J Clin Invest100:3164-72 (1997)). The regulation of gene expression by insulin in theliver is impaired for the genes for glucokinase and phosphoenolpyruvatecarboxykinase (PEPCK) (Friedman et al., J Biol Chem 272:31475-81(1997)). A modulator of insulin action is tumor necrosis factor(TNF)-α,which blocks insulin through its ability to inhibit insulin receptortyrosine kinase activity (Feinstein et al., J Biol Chem 268:26055-8(1993)). Mice lacking TNF-α function are protected from obesity-inducedinsulin resistance (Uysal et al., Nature 389:610-4 (1997)). Anothermodulator of insulin sensitivity is protein tyrosine phosphatase-1 B(PTP-1B) which acts as a negative regulator of insulin signaling(Cicirelli et al., Proc Natl Acad Sci USA 87:5514-8 (1990)). Micedeficient in PTP-1B are interestingly more sensitive to insulin butresistant to obesity (Elchebly et al., Science 283:1544-8 (1999)). Mostrecent studies have focused on the peroxisome proliferator-activatedreceptor γ (PPARγ), a member of the nuclear-hormone-receptor family(Auwerx, Diabetologia 42:1033-49 (1999)). Mutations in humans of PPARγsuggest that this molecule is required for normal insulin sensitivity inhumans (Barroso et al., Nature 402:880-3 (1999)). It is not clear at themoment whether insulin resistance in human obesity might result fromimpaired PPARγ signaling. What is now clear is that decreased signalingcapacity of the insulin receptor can be an important component ofobesity-induced insulin resistance.

[0006] At the intracellular, metabolic enzyme, level, insulin-resistancein obesity seems to consist of increased activities of key enzymes ofpathways known to be stimulated by insulin (i.e. glycolysis,lipogenesis), but also of increased activities of key enzymes ofpathways normally depressed by insulin (Belfiore et al., Int J Obes3:301-23 (1979)). This failure of insulin to depress enzymes ofcatabolic pathways manifests itself in enhanced basal lipolysis inadipose tissue, increased amino acid release from muscle, and elevationin the activity of key gluconeogenic enzymes in the liver.

[0007] As mentioned above, there are a number of mouse models withgenetic obesity-diabetes syndromes (Herberg, et al., Metabolism 26:59-99(1977)). They characteristically have hyperglycemia, hyperinsulinemia,and obesity, albeit to different degrees, with different times of onset,and for different reasons. In the yellow obese mouse (A^(y)/a) adominant mutation of the agouti locus causes the ectopic, ubiquitousexpression of the agouti protein, resulting in a condition similar toadult-onset obesity and non-insulin-dependent diabetes mellitus (Michaudet al., Proc Natl Acad Sci USA 91:2562-6 (1994)). Obese (ob/ob) (Zhanget al., Nature 372:425-32 (1994)), diabetes (db/db) (Tartaglia et al.,Cell 83:1263-71 (1995)), fat (cpe/cpe) (Naggert et al., Nat Genet10:135-42 (1995)) and tubby (tub/tub) (Kleyn et al., Cell 85:281-90(1996); Noben-Trauth et al., Nature 380:534-8 (1996)) are mutations insingle recessive genes, specifically in the genes for leptin, the leptinreceptor, carboxypeptidase E, and a member of a new family of genesencoding tubby-like proteins, respectively. Obese mice have adiabetes-like syndrome of hyperglycemia, glucose intolerance, andelevated plasma insulin. The diabetes syndrome develops after the onsetof obesity, and is probably the result of it. In diabetes mice elevationof plasma insulin at 2 weeks of age precedes the onset of obesity at 3-4weeks; blood glucose levels are elevated at 4-8 weeks. Fat mice havehyperinsulinemia consistent throughout life in association withhypertrophy and hyperplasia of the islets of Langerhans; hyperglycemiais transient. In tubby mice, plasma insulin is increased prior toobvious signs of obesity, and islets of Langerhans are enlarged; hereblood glucose is normal.

[0008] As discussed above, the molecular basis for insulin resistance inobesity is unknown. Increased leptin levels cannot account for this,since insulin resistance occurs in the leptin deficient ob/ob mutants.Therefore, there must be some other molecular “signal” in obesity whichmediates the insulin-resistance seen in obesity.

[0009] Faced with such a long felt, but unsolved need for simple andeffective methods to prevent or reduce the negative effects of diabetes,researchers, over the last several decades, have expended literallyhundreds of millions of dollars to investigate compounds that can beused to treat and/or prevent diabetes. While altering glucose can affectthe occurrence and the severity of diabetes, so can the regulation ofinsulin resistance in obesity. This latter approach has been anunder-appreciated field relative to diabetes. The present invention isdirected to the prevention and/or treatment of diabetes through theregulation of insulin resistance in obesity.

SUMMARY OF THE INVENTION

[0010] One embodiment of the present invention relates to a method toidentify compounds useful in regulating insulin resistance in obesityand type II diabetes. This method includes the steps of: (a)administering a compound having melanocyte stimulating hormone (MSH)biological activity to a genetically modified non-human animalcomprising a genetic modification within two alleles of its Pomc locus,wherein the genetic modification results in an absence ofproopiomelanocortin (Pomc) peptide activity in the animal, and whereinadministration of the compound having MSH activity induces insulinresistance in the animal; (b) administering a compound to be evaluatedto the non-human animal model; and, (c) selecting compounds from (b)that decrease the insulin resistance in the non-human animal as comparedto in the absence of the compound of (b).

[0011] In one embodiment, the genetic modification is selected from thegroup consisting of a deletion, an insertion, a substitution and aninversion of nucleotides in the Pomc locus.

[0012] In another embodiment, the genetic modification is a deletion ofa nucleic acid sequence within two alleles of the Pomc locus, whereinthe deletion results in an absence of expression of Pomc peptides by theanimal. In another embodiment, the genetic modification is a deletion ofa nucleic acid sequence comprising exon 3 of Pomc or a portion of exon 3of Pomc sufficient to prevent expression of Pomc peptides by two allelesof the Pomc locus. In another embodiment, the animal is a mouse, andwherein the genetic modification is a deletion from the genome of exon 3of Pomc (SEQ ID NO:7).

[0013] In one aspect, the compound having MSH biological activity instep (a) is selected from the group consisting of: MSH, a biologicallyactive fragment of MSH, a homologue of MSH, a peptide mimetic of MSH, anon-peptide mimetic of MSH, and a fusion protein comprising an MSHprotein or fragment thereof. In another aspect, the compound of (a)having MSH biological activity is α-MSH.

[0014] In one aspect, the compound of (b) to be evaluated is anantagonist of MSH biological activity. In another aspect, the compoundof (b) to be evaluated is administered prior to the step ofadministering the compound of (a) having MSH biological activity.

[0015] Yet another embodiment of the present invention relates to amethod to decrease insulin resistance in a mammal, comprisingadministering to the mammal that has insulin resistance a therapeuticcomposition comprising an antagonist of melanocortin stimulating hormone(MSH) biological activity, wherein the antagonist decreases insulinresistance in the mammal. In one aspect, the antagonist of melanocortinstimulating hormone (MSH) is selected from the group consisting of afragment of MSH having MSH antagonist action, a homologue of MSH havingMSH antagonist action, a peptide mimetic of MSH having MSH antagonistaction, a non-peptide mimetic of MSH having MSH antagonist action, and afusion protein comprising any of the MSH antagonist compounds. Inanother aspect, the antagonist of MSH is a soluble MSH receptor orfragment thereof that binds MSH. In yet another aspect, the antagonistof MSH is an antibody that selectively binds to MSH and thereby reducesor blocks the activity of MSH. In another aspect, the antagonist of MSHis an antibody that selectively binds to a receptor for MSH and reducesor blocks the ability of MSH to bind to the receptor.

[0016] The therapeutic composition can be administered by any suitableroute, including, but not limited to, transdermally, topically, andparenterally. In one aspect, the therapeutic composition is administeredin a controlled release formulation. In one aspect, the MSH antagonistis administered in a dose of from about 0.1 μg to about 10 mg per kgbody weight of the animal.

[0017] Another embodiment of the present invention relates to a methodto treat diabetes associated with insulin resistance in a mammal,comprising administering to the mammal that has insulin resistance anddiabetes a therapeutic composition comprising an antagonist ofmelanocortin stimulating hormone (MSH) biological activity, wherein theantagonist decreases insulin resistance in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

[0018]FIG. 1A is a schematic diagrams and restriction map of the mousePomc locus, the targeting vector, and the predicted structure of thePomc locus after homologous recombination.

[0019]FIG. 1B is a scanned image of a Southern blot analysis of tailDNAs from F₂ littermates.

[0020]FIG. 1C is a bar graph showing an RIA analysis of serum ACTHlevels in F₂ male littermates.

[0021]FIG. 2A is a line graph of weight measurements taken from malemice of wildtype and mutant POMC genotype.

[0022]FIG. 2B is a bar graph illustrating that mutant POMC mice showincreased linear growth.

[0023]FIG. 2C is a bar graph illustrating that POMC null mice haveelevated leptin serum levels.

[0024]FIG. 2D is a bar graph illustrating weight change for POMC nullmice and wildtype mice being fed a standard diet or a high fat diet.

[0025]FIG. 2E is a bar graph illustrating food intake for POMC null miceand wildtype mice being fed a standard diet or a high fat diet.

[0026]FIG. 3A is a bar graph showing that corticosterone levels inmutant POMC mice were below the detection limit of the RIA.

[0027]FIG. 3B is a bar graph showing that aldosterone levels in mutantPOMC mice were below the detection limit of the RIA.

[0028]FIG. 3C is a bar graph showing that epinephrine levels weresignificantly lower in mutant POMC mice as compared to wildtype mice.

[0029]FIG. 3D is a bar graph showing that norepinephrine levels were notsignificantly different in mutant POMC mice as compared to wildtypemice.

[0030]FIG. 3E is a bar graph showing that dopamine levels were slightlyincreased in mutant POMC mice as compared to wildtype mice.

[0031]FIG. 4A is a bar graph showing the blood glucose levels in POMCnull mutant mice and controls in the fed state.

[0032]FIG. 4B is a bar graph showing the insulin levels in POMC nullmutant mice and controls in the fed state.

[0033]FIG. 4C is a bar graph showing the blood glucose levels in POMCnull mutant mice and controls in the fasting state.

[0034]FIG. 4D is a bar graph showing the insulin levels in POMC nullmutant mice and controls in the fasting state.

[0035]FIG. 5 is a line graph showing the blood glucose levels of POMCnull mutants over time.

[0036]FIG. 6 is a line graph showing the blood glucose levels during aninsulin tolerance test with POMC null mutants.

[0037]FIG. 7 is a bar graph showing the effects of corticosteronetreatment on blood glucose in POMC null mutants.

[0038]FIGS. 8A and 8B are line graphs showing the effects ofcorticosterone treatment on blood glucose in an insulin tolerance testwith POMC null mutants.

[0039]FIG. 9 is a line graph showing the effects of MSH and ACTHtreatment on blood glucose in an insulin tolerance test with POMC nullmutants.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention generally relates to methods foridentifying compounds the decrease insulin-resistance associated withobesity and diabetes, and to methods of decreasing insulin-resistance ina mammal in need of such treatment. The present invention is alsodirected to the treatment of diabetes associated withinsulin-resistance. The invention is predicated upon the presentinventors' surprising discovery that a non-human animal model forobesity, in contrast to previously described animal models for obesity,is protected from obesity-induced insulin resistance and diabetes. Giventhe close relationship between obesity and diabetes, an obesity mutantwhich is resistant to diabetes represents a unique disconnection betweenthe two conditions. More specifically, the present inventors havecreated a genetic mouse model of obesity, the POMC null mouse (Yaswen etal., Nat Med 5:1066-1070 (1999)), which the present inventors havesurprisingly found is protected from obesity-induced type 2 diabetes andis sensitive to insulin. The POMC null mouse has been previouslydescribed in U.S. patent application Ser. No. 09/374,827, filed Aug. 12,1999, entitled, “Non-Human Animal Model for Obesity and Uses Thereof”,in U.S. Provisional Application Serial No. 60/111,581, filed Dec. 9,1998, entitled, “Composition and Method for Controlling Obesity”, and inU.S. Provisional Application Serial No. 60/146,306, filed Jul. 29, 1999,entitled “Composition and Method for Controlling Body Weight andConditions Related Thereto”, each of which is incorporated herein byreference in its entirety. However, prior to the present invention, itwas not known that the POMC null mutant mouse would be neitherinsulin-resistant nor diabetes prone, and as mentioned above, such adiscovery was surprising. The present inventors have additionally foundthat administration of a melanocortin stimulating hormone (MSH) to thePOMC null mouse induces insulin-resistance in the mouse, indicating thatit is the lack of MSH in the mouse model that protects the animal frominsulin-resistance. This mouse model can now be used to investigate thebiochemical and molecular mechanisms associated with insulin-resistanceand diabetes, and to identify compounds that reduce insulin-resistancein an animal and/or reduce the symptoms of type II diabetes. Moreover,the present inventors have provided evidence that indicates thatantagonizing the biological activity of MSH can be used to reduceinsulin resistance in an individual, which in turn would be beneficialin the treatment of diabetes associated with insulin resistance. Indeed,subsequent to the present invention, Katsuki et al. showed that elevatedplasma levels of α-MSH are correlated with insulin resistance in obesemen (Katsuki et al., October 2000, Int. J. Obesity 24:1260-1264).

[0041] As discussed above, the present inventors have disclosed thedevelopment and characterization of a Pomc mutant mouse which is a modelof obesity. This model has now surprisingly been found to beinsulin-sensitive, rather than insulin-resistant as are other obesemouse models, thus providing a useful animal model for dissecting thefactors that contribute to obesity and insulin-resistance. The Pomcmutant mouse was engineered to carry an autosomal recessive null alleleof the Pomc gene (i.e., pomc). This mouse lacks all of the peptidehormones encoded by the Pomc locus. The present inventors havediscovered that mice lacking the Pomc peptides have obesity, a defect inadrenal development, and altered pigmentation. This phenotype is similarto the recently identified human Pomc mutants (Krude, et al., 1998, NatGenet 19,155-7). In addition to a dysregulation of fat metabolism, thePOMC-deficient mice showed increased food intake.

[0042] When the present inventors treated the mutant mice peripherallywith a stable α-MSH agonist, these mice lost over 40% of their excessweight after two weeks, whereas wildtype non-obese mice did not losesignificant weight. The present inventors have shown that the weightchanges in POMC null mice are not simply regulated through feedingbehavior, but rather through both central and peripheral actions ofmelanocortins.

[0043] The genetically modified non-human animal useful in the presentinvention comprises a genetic modification within at least one allele ofits Pomc locus, wherein the genetic modification results in a reductionin proopiomelanocortin (Pomc) peptide action (activity) in the animal(e.g., a heterozygous mutant animal). In one embodiment, the geneticmodification includes, but is not limited to, a deletion, an insertion,a substitution and/or an inversion of nucleotides in the Pomc locuswhich result in a reduction in Pomc peptide action in the animal. Thegenetic modification can be a modification including or within exon 3 ofthe Pomc locus which results in a reduction in Pomc peptide action,and/or a modification in a region of the Pomc locus other than exon 3which results in a reduction in Pomc peptide action (e.g., exon 1, exon2 and/or a regulatory region of the Pomc locus). In a preferredembodiment, the genetic modification is a deletion of a nucleic acidsequence within at least one allele of the Pomc locus, wherein thedeletion results in an reduction of expression of Pomc peptides by saidanimal. In another embodiment, the animal comprises a geneticmodification within two alleles (i.e., both alleles) of the Pomc locus,wherein the genetic modification results in an absence of Pomc peptideaction in the animal (e.g., a homozygous mutant animal). Preferably, thegenetic modification is a deletion of a nucleic acid sequence withinboth alleles of the Pomc locus, wherein the deletion results in anabsence of expression of Pomc peptides by the animal.

[0044] Proopiomelanocortin (Pomc) peptides, including the melanocortins:adrenocorticotrophin (ACTH); α-, β- and γ-melanocyte stimulatinghormones (MSH); and the opioid receptor ligand β-endorphin, have adiverse array of biological activities, including roles in pigmentation,adrenocortical function, regulation of energy stores, and the immune,central nervous and peripheral circulation system (Smith, A. I. et al.,Endocr Rev 9, 159-179 (1988); König, “Peptide and protein hormones:structure, regulation, activity, a reference manual” (Weinheim; NY1993)). As used herein, reference to Pomc peptides is intended to refergenerically to any one or more of the Pomc peptides encoded by the Pomclocus. If reference to a specific Pomc peptide, such as MSH, isintended, the name of the specific peptide will be used. The nucleicacid and amino acid sequences for the naturally occurring Pomc peptidesin a large variety of animals (i.e., human, mouse, rat, rabbit, bovine,ovine, macaque, amphibian, etc.) are known in the art. Such sequencescan be found, for example, in a protein or nucleic acid database such asGenBank. GenBank accession numbers for such Pomc peptide (i.e., aminoacid) sequences include, but are not limited to: Accession Nos.NP_(—)000930 or CAA24754 (Homo sapiens); Accession No. P06297 (rabbit);Accession No. P01194 (rat); Accession No. P01193 (mouse); Accession No.P01191 (sheep); and, Accession No. P01190 (bovine). GenBank accessionnumbers for such Pomc nucleic acid sequences include, but are notlimited to: Accession No. NM_(—)000939 (Homo sapiens); Accession No.AH005319 (mouse); Accession Nos. J00016, J00019, J00021 (bovine);Accession No. S73519 (swine); S57982 (ovine); and Accession No. AH002232(rat). Exons 1, 2 and 3 for the mouse Pomc locus are identified asGenBank Accession Nos. J00610, J00611 and J00612, respectively.

[0045] As used herein, a non-human animal suitable for geneticmodification and use according to the present invention is any non-humananimal for which the Pomc locus can be manipulated, including non-humanmembers of the Vertebrate class, Mammalia, such as non-human primatesand rodents. Preferably, such a non-human animal is a rodent, and morepreferably, a mouse. Genetically modified mice which have either areduction or an absence of Pomc peptide expression are described indetail in the Examples section (e.g., see Example 1).

[0046] According to the present invention, a “genetically modified”animal, such as any of the preferred non-human animals described herein,has a genome which is modified (i.e., mutated or changed) from itsnormal (i.e., wild-type or naturally occurring) form such that thedesired result is achieved (e.g., a reduction in the action of Pomcpeptides). Genetic modification of an animal is typically accomplishedusing molecular genetic and cellular techniques, including manipulationof embryonic cells and DNA (e.g., DNA comprising the Pomc locus). Suchtechniques are generally disclosed for mice, for example, in“Manipulating the Mouse Embryo” (Hogan et al., Cold Spring HarborLaboratory Press, 1994, incorporated herein by reference in itsentirety). Additionally, techniques for genetic modification of a mousethrough molecular technology are described in detail in the Examplessection.

[0047] A genetically modified non-human animal can include a non-humananimal in which nucleic acid molecules have been modified (i.e.,mutated; e.g., by insertion, deletion, substitution, and/or inversion ofnucleotides), in such a manner that such modifications provide thedesired effect within the animal (i.e., reduction in Pomc peptideaction). As used herein, genetic modifications which result in areduction in gene expression, in the function of the gene, or in thefunction of the gene product (i.e., the protein encoded by the gene) canbe referred to as inactivation (complete or partial), deletion,interruption, blockage or down-regulation of a gene. For example, agenetic modification in a gene which results in a decrease in thefunction of the protein encoded by such gene, can be the result of: apartial or complete deletion of the gene or of an exon within the gene(i.e., the gene does not exist, and therefore the protein can not beproduced); a mutation (e.g., a deletion, substitution, insertion and/orinversion) in the gene which results in incomplete or no translation ofthe protein (e.g., a mutation which causes a frame shift so that thecorrect protein is not expressed, a mutation in one or more exons of thegene so that the protein or at least a portion of the protein is notexpressed, or a mutation in a regulatory region so that the protein isnot expressed or has reduced expression); or a mutation in the genewhich decreases or abolishes the natural function of the protein (e.g.,a protein is expressed which has decreased or no biological activity oraction).

[0048] According to the present invention, a genetic modification of anon-human animal results in a reduction (i.e., decrease, inhibition,down-regulation) of the action of Pomc peptides. Such a geneticmodification includes any type of modification to a genome of theanimal, particularly including modifications made at the embryonic stageof development of the animal (or in the ancestor of the animal). Suchmodifications are described above. According to the present invention,reference to reducing “the action” (or activity) of Pomc peptides refersto any genetic modification in the non-human animal which results indecreased functionality of one or more of the Pomc peptides, including:reduced biological activity of the peptides (e.g., reduced in vivohormonal activity); inhibition or degradation of the peptides (i.e., thepeptides are expressed, but are inhibited or degraded as a result of thegenetic modification); and reduced, or abolished, expression of thepeptides (i.e., by complete or partial gene deletion, substitution,insertion, etc.). For example, the action of Pomc peptides can bedecreased by blocking or reducing the production of the peptides,“knocking out” the gene or a portion of the gene encoding the peptides,reducing peptide activity, or inhibiting the activity of the peptides.

[0049] In one embodiment of the present invention, a non-human animal ofthe present invention is genetically modified by modification of anucleic acid sequence within one (i.e., heterozygous) or both (i.e.,homozygous) alleles of the Pomc locus, wherein such modification caninclude, but is not limited to, a deletion, an insertion, a substitutionand/or an inversion within the one or more nucleotides in the Pomclocus. In one embodiment, the genetic modification is in a nucleic acidsequence that includes exon 3 of the Pomc locus, such modificationresulting in a decrease in Pomc peptide action in the animal. In anotherembodiment, the genetic modification is in a region of the Pomc locusother than exon 3, whereby the modification results in a decrease inPomc peptide action in the animal. Such other regions include exon 1,exon 2 or a regulatory region of the Pomc locus. According to thepresent invention, a regulatory region of a gene includes any regulatorysequences that control the expression of nucleic acid molecules,including promoters, enhancers, transcription termination sequences,sequences that regulate translation, and origins of replication.

[0050] In a preferred embodiment of the present invention, a non-humananimal of the present invention is genetically modified by deletion of anucleic acid sequence within one or both alleles of the Pomc locus,wherein the deletion results in a reduction or absence, respectively, ofexpression of Pomc peptides by the animal. An animal having amodification in both alleles of the Pomc locus such that themodification results in the absence of Pomc activity, can be referred toas a null mutant or POMC null mutant. In one embodiment, such a geneticmodification is a deletion of a nucleic acid sequence comprising exon 3of Pomc. In another embodiment, the genetic modification is a deletionof exon 3 of Pomc. In yet another embodiment, the genetic modificationis a deletion of a portion of exon 3 of Pomc sufficient to reduce orprevent expression of Pomc peptides by at least one allele and morepreferably, by both alleles, of the Pomc locus of the animal.

[0051] In one embodiment of the present invention, the geneticallymodified non-human animal is a mouse, also referred to herein as a POMChomozygous mutant mouse or POMC null mutant mouse. In this embodiment,the genetic modification is preferably a deletion from the genome of anucleic acid sequence comprising SEQ ID NO:7, although any geneticmodification of the Pomc locus as described above is encompassed by thepresent invention. SEQ ID NO:7 represents exon 3 of the mouse (i.e., Musmusculus) Pomc locus and can be located in the GenBank database asGenBank Accession No. J00612. SEQ ID NO:7 encodes an amino acid sequencerepresented herein as SEQ ID NO:8. Preferably, the genetic modificationin the mouse is a deletion from the genome of exon 3 of Pomc (SEQ IDNO:7).

[0052] The genetically modified non-human animal of the presentinvention can be characterized by several phenotypes which result fromthe reduction or absence in Pomc peptide action in the animal. Suchphenotypic characteristics include: obesity, a defect in adrenaldevelopment, and/or altered pigmentation. In addition, the presentinventors have discovered that such a genetically modified non-humananimal has measurably increased serum leptin levels as compared to awild-type sibling of the animal. Other phenotypic characteristicsassociated with the genetic modification include: an increased fooduptake as compared to a wild-type sibling of the animal and/ormeasurably reduced serum levels of a hormone selected from the group ofcorticosterone, aldosterone and epinephrine as compared to a wild-typesibling of the animal. Most importantly in the present invention,phenotypic characteristics associated with the genetic modification alsoinclude normal glucose and insulin levels over the life of the animaland an inhibited glucoregulatory response during an insulin tolerancetest.

[0053] As used herein, a wild-type sibling, or wild-type littermate, isan animal which is born to the same or genetically identical parents asa genetically modified animal described herein, and preferably, is bornin the same litter as a genetically modified animal described herein,but which did not inherit a genetically modified allele at the Pomclocus. Such an animal is essentially a normal animal and is useful as anage-matched control for the methods described herein.

[0054] According to the present invention, a non-human animal can begenetically modified by any method which results in the desired effect(i.e., reduction in Pomc peptide action in the animal). Such methods aretypically molecular techniques, and include, but are not limited to, anydeletion of at least a portion of the Pomc locus in the animal, anyinsertion of a non-Pomc sequence into at least a portion of the Pomclocus in the animal, or any substitution of at least a portion of thePomc locus in the animal with any non-Pomc sequence or mutated Pomcsequence, sufficient to reduce Pomc peptide action in the animal. Forexample, a Pomc locus in the genome of an animal (or an embryonic cell)can be genetically modified by inserting into at least one allele of thePomc locus of the animal or cell an isolated nucleic acid molecule whichencodes at least a section of the Pomc gene. At least a portion of thisisolated section of the Pomc gene is mutated (i.e., by deletion of theportion, substitution of the portion with another, non-Pomc sequence, orinsertion of a non-Pomc sequence into the section of Pomc), such thatwhen the isolated nucleic acid molecule is inserted into the endogenousPomc locus of the animal or cell, the animal or cell will have areduction or elimination in the action of Pomc peptides as describedabove. As another example, in one embodiment of the invention, agenetically modified mouse is produced by inserting into the genome ofan embryonic stem (ES) cell an isolated nucleic acid molecule (e.g., atargeting vector) having an isolated nucleic acid sequence encoding themurine Pomc gene. In this isolated nucleic acid sequence, exon 3 of themurine Pomc gene has been deleted and replaced with a non-Pomc nucleicacid sequence (e.g., a marker sequence, such as a neomycin cassette).The isolated nucleic acid molecule is preferably designed such that whenthe molecule is injected into embryonic stem (ES) cells, the isolatednucleic acid molecule will integrate into the genome of the cells,preferably at the endogenous Pomc locus (i.e., targeted integration).

[0055] Techniques for achieving targeted integration of an isolatednucleic acid molecule into a genome are well known in the art and aredescribed, for example in “Manipulating the Mouse Embryo”, supra. Forexample, the isolated nucleic acid molecule can be engineered into atargeting vector which is designed to integrate into a host genome.According to the present invention, a targeting vector is defined as anucleic acid molecule which has the following three features: (1)genomic sequence from the target locus in the host genome to stimulatehomologous recombination at that locus; (2) a desired geneticmodification within the genomic sequence from the target locussufficient to obtain the desired phenotype; and (3) a selectable marker(e.g., an antibiotic resistance cassette, such as G418, neomycin, orhygromycin resistance cassettes). Such targeting vectors are well knownin the art. Following introduction of the isolated nucleic acid moleculeof the targeting vector into the ES cells, ES cells which homologouslyintegrate the isolated nucleic acid molecule are injected into mouseblastocysts and chimeric mice are produced. These mice are then bredonto the desired mouse background to detect those which transmit themutated gene through the germ line. Heterozygous offspring of germlinetransmitting lines can then be mated to produce homozygous progeny.

[0056] Mice which carry one or more mutated Pomc alleles can beidentified using any suitable method for evaluating DNA. For example,genotypes can be analyzed by PCR and confirmed by Southern blot analysisas described (Sambrook et al., 1988, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. or Current Protocols in Molecular Biology (1989) andsupplements).

[0057] According to the present invention, an isolated nucleic acidmolecule suitable for use in the present invention (e.g., suitable foruse in a targeting vector according to the invention) is typicallyproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. DNAcomprising the desired nucleic acid sequence (e.g., the Pomc locus,modified or unmodified) may be created, for example, by using polymerasechain reaction (PCR) techniques or other cloning techniques. Thetemplate can be a genomic or cDNA library isolated from central nervoussystem or pituitary tissue. Such methodologies are well known in the art(Sambrook et al., supra).

[0058] Isolated nucleic acid molecules useful in the present inventioncan be modified by nucleotide insertions, deletions, and substitutions(e.g., nucleic acid homologues) in a manner such that the modificationsproduce the desired effect (e.g., a deletion or substitution of aportion of a Pomc gene sufficient to reduce POMC action in an animalwhen the nucleic acid molecule is integrated into the animal's genome).An isolated nucleic acid molecule encoding Pomc peptides can includedegeneracies. As used herein, nucleotide degeneracies refers to thephenomenon that one amino acid can be encoded by different nucleotidecodons. Thus, the nucleic acid sequence of a nucleic acid molecule thatencodes a Pomc peptide of the present invention can vary due todegeneracies.

[0059] One embodiment of the present invention relates to a method toidentify compounds, and particularly antagonists of MSH biologicalactivity, for use in regulating insulin resistance in obesity andparticularly, in non-insulin dependent diabetes mellitus (NIDDM) or typeII diabetes. Such a method includes screening a compound to be evaluatedfor its ability to decrease insulin resistance in a genetically modifiednon-human animal of the present invention (i.e., a POMC null mutant) inwhich insulin resistance has been induced. Compounds identified by sucha method may then be useful in a method to reduce insulin resistance ina patient, which is another embodiment of the present invention. Such amethod can be performed in vitro (e.g., by using cells, tissues or bodyfluids of the genetically modified animal) or in vivo (e.g., byadministering regulatory compounds to a genetically modified animal ofthe present invention and evaluating the effects of such compounds invivo). Regulatory compounds identified by this method are useful forinhibiting insulin resistance in an animal that has insulin resistance(e.g., an obese animal or a type II diabetic animal), and may haveadditional beneficial therapeutic effects on disorders and conditionsrelated to excess body weight in an animal.

[0060] In one aspect, such a method includes the steps of: (a)harvesting cells, tissues or body fluids from a genetically modifiednon-human animal which comprises a genetic modification within two(both) alleles of its Pomc locus, wherein the genetic modificationresults in an absence of Pomc peptide activity in the animal; and, (b)comparing the cells, tissues or body fluids from the geneticallymodified non-human animal to cells, tissues or body fluids from awild-type sibling of the genetically modified non-human animal in thepresence and absence of a compound to be evaluated for its ability toregulate insulin resistance, or after administration of a compound to beevaluated to the animal. The step of harvesting is performed using anyof the well known methods of harvesting cells, tissues and/or bodyfluids from an animal, and depend on the tissues to be studied and thestatus of the experiment to be performed. For example, cells can beharvested by biopsy, dissection, or lavage; tissues can be harvested bysurgery, biopsy or dissection; and body fluids can be harvested bywithdrawal, swiping, or lavage.

[0061] The step of comparing is performed by an assay that is suitablefor the tissue to be evaluated and the goal of the experiment. Forexample, suitable assays which might be performed on the cells, tissues,and/or body fluids of a genetically modified non-human animal of thepresent invention include, but are not limited to: morphologicalexamination of the cells, tissues or body fluids; histologicalexamination of the cells, tissues or body fluids; evaluation of Pomcpeptide biological activity in the animal; evaluation of free fatty acidmetabolism in the animal; evaluation of lipolysis and fatty acidsequestration in the animal; evaluation of insulin, glucagon and glucoselevels; evaluation of weight gain or loss in the animal; evaluation ofhormone levels in the animal; evaluation of blood biochemistry in theanimal. A variety of such assays are well known in the art.

[0062] In another aspect, the method of the present invention includesthe steps of: (a) administering a compound having melanocortinstimulating hormone (MSH) biological activity to a genetically modifiednon-human animal which comprises a genetic modification within two(both) alleles of its Pomc locus, wherein the genetic modificationresults in an absence of Pomc peptide activity in the animal, andwherein the administration of the compound induces insulin resistance inthe animal in the absence of any other opposing factor; (b)administering a compound to be evaluated to the animal, either prior to,simultaneous with, or after administration of the compound having MSHbiological activity of (a); and, (c) selecting compounds in (b) whichreduce insulin resistance in the animal, as compared to in the absenceof the administration of the compound in (b).

[0063] One step of the method of the present invention includes the stepof administering to the genetically modified non-human animal that is ahomozygous Pomc mutant a compound that has melanocyte stimulatinghormone (MSH) biological activity, wherein administration of such acompound induces insulin resistance in said animal. According to thepresent invention, in general, the biological activity or biologicalaction of a protein such as an MSH peptide or homologue or mimeticthereof refers to any function(s) exhibited or performed by the protein(or mimetic) that is ascribed to the naturally occurring form of theprotein as measured or observed in vivo (i.e., in the naturalphysiological environment of the protein) or in vitro (i.e., underlaboratory conditions). In particular, the biological activity of MSHthat is of interest herein includes the ability of MSH to induce insulinresistance in a POMC null mutant mouse.

[0064] According to the present invention, “insulin resistance”, whichcan also be described as a reduction in insulin sensitivity, refers to areduced sensitivity in the tissues of the body to the action of insulin,as compared to a previous, “predicted” or “normal” value for insulinaction. More specifically, insulin resistance is defined as an impairedbiological response to either exogenous or endogenous insulin. Wheninsulin resistance, or reduced insulin sensitivity, exists, the bodyattempts to overcome this resistance by secreting more insulin from thepancreas. This compensatory state of hyperinsulinemia (high insulinlevels in the blood) can be used as a marker for the existence ofinsulin resistance. The high insulin levels resulting from insulinresistance contribute to abnormalities in blood lipids, includingcholesterol and triglycerides.

[0065] A variety of procedures have been developed to detect thepresence of insulin resistance. Such procedures include, but are notlimited to, measurement by the euglycemic insulin clamp, measurement bythe minimal model, and measurement of the fasting insulin level.Briefly, in the euglycemic insulin clamp method, exogenous insulin isinfused, so as to maintain a constant plasma insulin level abovefasting, while glucose is fixed at a basal level by infusing glucose atvarying rates. This glucose infusion is delivered via an indwellingcatheter at a rate based on plasma glucose measurements every 5 min.When the plasma glucose level falls below basal, the glucose infusionrate is increased to return plasma glucose to basal levels and viceversa. The total amount of glucose infused over time (M value) is anindex of insulin action on glucose metabolism. The more glucose that hasto be infused per unit time, then the more sensitive the patient is toinsulin. Conversely, the insulin-resistant patient requires much lessglucose to maintain basal plasma glucose levels.

[0066] In the minimal model, glucose and insulin are sampled frequentlyfrom an indwelling catheter during an intravenous glucose tolerancetest; the results are entered into a computer model, which generates avalue that is an index of insulin sensitivity (called S_(i)). The acuteinsulin release (AIR) in response to glucose is also determined by thetest.

[0067] The measurement of fasting insulin level is typically performedin the overnight fasted condition. There is a significant correlationbetween fasting insulin levels and insulin action as measured by theclamp technique. Moreover, it is generally true that very high plasmainsulin values in the setting of normal glucose levels are very likelyto reflect insulin resistance.

[0068] Therefore, according to the present method, an increase ininsulin resistance (e.g., a decrease in insulin sensitivity) or adecrease in insulin resistance (an increase in insulin sensitivity),refers to a change in the ability of an animal to respond to insulin ascompared to a previous measure, to a general control measure that hasbeen established for that animal, or to a predicted “normal” measurethat has been established for that animal. An increase or decrease ininsulin resistance or sensitivity can be any detectable change inresponse to insulin as determined by any suitable method of measuringinsulin sensitivity/resistance.

[0069] Preferably, compounds having MSH biological activity (e.g., MSHand MSH agonists) are any compound having one or more of the followingproperties or identifying characteristics: (1) an ability to bind to anMSH receptor; and, (2) an ability to stimulate lipolysis and/or toinhibit the uptake of fatty acids by adipocytes. Particularly preferredMSH compounds for use in step (a) of the present method includehomologues and mimetics of naturally occurring MSH peptides which havesubstantially similar, or even more preferably, enhanced, properties oridentifying characteristics as compared to the naturally occurring(i.e., prototype) MSH (e.g., agonists). Such properties or identifyingcharacteristics can include: (1) enhanced ability to bind to a MSHpeptide receptor; (2) enhanced serum half-life (i.e., enhanced stabilityunder physiological conditions); and/or (3) enhanced ability tostimulate lipolysis and/or to inhibit the uptake of fatty acids byadipocytes.

[0070] In one embodiment, the compound having MSH biological activitycan include any peptide that has an amino acid sequence which includesthe amino acid sequence represented herein by SEQ ID NO: 1 (EHFRW), or ahomologue or mimetic thereof. In another embodiment, a preferredcompound having MSH biological activity includes, but is not limited to,a melanocortin stimulating hormone peptide, fragments of such peptides,fusion proteins comprising such peptides, and any MSH agonist, includinghomologues of MSH peptides, mimetics (peptide or non-peptide) of suchpeptides, and any pharmaceutical salts of such peptides. Preferredmelanocyte stimulating hormones (MSH) include α-MSH, β-MSH and γ-MSH,fragments of such peptides, homologues of such peptides, mimetics(peptide or non-peptide) of such peptides, fusion proteins comprisingsuch peptides, and any pharmaceutical salts of such peptides.

[0071] The amino acid sequence of human A-MSH is:

[0072] Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂;

[0073] and is represented herein by SEQ ID NO:2. It should be noted thatsince amino acid sequencing and nucleic acid sequencing technologies arenot entirely error-free, any sequences presented or referenced herein,at best, represent apparent sequences of MSH peptides, homologues,peptide mimetics, and nucleic acid sequences encoding such peptides,useful in the present invention.

[0074] Another step of the method of the present invention, which can beperformed prior to, simultaneously with, or after the step ofadministering the compound having MSH biological activity, includes thestep of administering to the genetically modified non-human animal aregulatory compound to be evaluated. According to the present invention,suitable compounds to be evaluated for regulatory activity in thepresent method preferably include compounds which have an unknownregulatory activity or an undetermined level of regulator activity, atleast with respect to the ability of such compounds to regulate insulinresistance. Particularly preferred putative regulatory compounds to testin the method of the present invention include any antagonist of MSHbiological activity, and can include a homologue of a MSH with MSHantagonist activity, a peptide or non-peptide mimetic of MSH with MSHantagonist activity, a fusion protein including an MSH antagonistpeptide, or a recombinant nucleic acid molecule encoding such a peptide,fragment, homologue, peptide mimetic, or fusion protein thereof. Asuitable regulatory compound can also include any compound, such as asmall molecule or drug that has MSH antagonist activity (i.e., it neednot necessarily be structurally similar to MSH).

[0075] According to the present invention, the term “compound”encompasses any of the following compounds: a peptide, a fragment of aknown peptide (including both biologically active and inactivefragments), a homologue of such a peptide, a mimetic (peptide ornon-peptide) of such a peptide, a fusion protein comprising such apeptide, and any pharmaceutical salts of such a peptide, as well as anysmall molecule or drug. In addition, peptides useful as regulatorycompounds in the present invention may exist, particularly whenformulated, as dimers, trimers, tetramers, and other multimers. Suchmultimers are included within the scope of the present invention. Asused herein, the term “analog”, as used in connection with a Pomcpeptide according to the present invention, refers generically to anyhomologue or mimetic (peptide or non-peptide) of a Pomc peptide. Termsused herein in connection with Pomc genes and proteins (e.g.,“compound”, “analog”, “homologue”, “mimetic”) can be similarly used withspecific Pomc genes and proteins (e.g., an MSH peptide, an MSH compound,an MSH analog, etc.). Homologues and mimetics are described in detailbelow. Analogs can include both agonists and antagonists of theprototype Pomc peptide.

[0076] As used herein, the phrase “MSH agonist ligand” or “MSH agonist”refers to any compound that interacts with an MSH receptor (i.e., areceptor that naturally binds to MSH, such as under physiologicalconditions) and that elicits an observable response. More particularly,an MSH agonist can include, but is not limited to, a protein, a peptide,a nucleic acid, an antibody or antigen-binding fragment thereof, acarbohydrate-based compound, a lipid-based compound, a natural organiccompound, a synthetically derived organic compound, or other compound(e.g., any product of drug design) that selectively binds to andactivates or increases the activation of an MSH receptor; and mostcommonly includes a homologue or mimetic of MSH, including a syntheticMSH which is characterized by its ability to agonize (e.g., stimulate,induce, increase, enhance) the biological activity of a naturallyoccurring MSH receptor in a manner similar to the natural agonist, MSH(e.g., by interaction/binding with and/or direct or indirect activationof an MSH receptor). The action of an agonist on one MSH receptor mayhave undesirable consequences in one tissue type and beneficialconsequences in another tissue type. However, the term agonist isintended to refer to the ability of the ligand to act on an MSH receptorin a manner that is substantially similar to the action of the naturalMSH ligand, MSH, on the MSH receptor.

[0077] The phrase, “MSH antagonist ligand” or “MSH antagonist” refers toany compound which inhibits the effect of an MSH agonist, as describedabove. More particularly, an MSH antagonist is capable of associatingwith an MSH receptor such that the biological activity of the receptoris decreased (e.g., reduced, inhibited, blocked, reversed, altered) in amanner that is antagonistic (e.g., against, a reversal of, contrary to)to the action of the natural agonist, MSH, on the receptor. An MSHantagonist can also act directly on an MSH agonist (e.g., MSH) to reduceor block the ability of MSH to bind to and activate its receptor, or tocause the elimination of MSH. Such a compound can include, but is notlimited to, a protein, a peptide, a nucleic acid, an antibody orantigen-binding fragment thereof, a carbohydrate-based compound, alipid-based compound, a natural organic compound, a syntheticallyderived organic compound, a soluble MSH receptor, or other compound(e.g., any product of drug design) that selectively binds to and blocksaccess to the receptor by a natural or synthetic agonist ligand (e.g.,by binding to either the receptor or a natural or synthetic agonist ofthe receptor) or reduces or inhibits the activity of an MSH receptor; ora product of drug design that blocks the receptor or alters thebiological activity of the receptor. In general, the term antagonist isintended to refer to the ability of the ligand to act on an MSH receptor(or MSH) in a manner that is antagonistic to the action of the naturalMSH ligand, MSH, or a synthetic MSH agonist or MSH homologue, on the MSHreceptor.

[0078] According to the present invention, agonists and antagonistligands can include any regulatory ligand or compound that has theabove-mentioned characteristics with regard to regulation of an MSHreceptor (i.e., any MSH receptor including, but not limited to MC1-R,MC2-R, MC3-R, MC4-R and MC5-R). An agonist can be strong or weak withmany levels in between. An antagonist can also be strong or weak. Someantagonists may have “mixed” agonist/antagonist properties.

[0079] As used herein, the term “homologue” is used to refer to apeptide which differs from a naturally occurring peptide (i.e., the“prototype”) by minor modifications to the naturally occurring peptide,but which maintains the basic peptide and side chain structure of thenaturally occurring form. Such changes include, but are not limited to:changes in one or a few amino acid side chains; changes one or a fewamino acids, including deletions (e.g., a truncated version of thepeptide) insertions and/or substitutions; changes in stereochemistry ofone or a few atoms; and/or minor derivatizations, including but notlimited to: methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. Preferably, a homologue has eitherenhanced or substantially similar properties compared to the naturallyoccurring peptide as discussed above (i.e., agonists), although peptideswith properties that antagonize the activity of the natural peptide(i.e., antagonists) are also encompassed by certain embodiments of thepresent invention.

[0080] Homologues of Pomc peptides (e.g., homologues of MSH) can be theresult of natural allelic variation or natural mutation. A naturallyoccurring allelic variant of a nucleic acid encoding Pomc peptide (or aprotein comprising an Pomc peptide) is a gene that occurs at essentiallythe same locus (or loci) in the genome as the gene which encodes suchPomc peptide, but which, due to natural variations caused by, forexample, mutation or recombination, has a similar but not identicalsequence. Allelic variants typically encode proteins having similaractivity to that of the protein encoded by the gene to which they arebeing compared. One class of allelic variants can encode the sameprotein but have different nucleic acid sequences due to the degeneracyof the genetic code. Allelic variants can also comprise alterations inthe 5′ or 3′ untranslated regions of the gene (e.g., in regulatorycontrol regions). Allelic variants are well known to those skilled inthe art.

[0081] Homologues can be produced using techniques known in the art forthe production of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

[0082] A mimetic refers to any peptide or non-peptide compound that isable to mimic the biological action of a naturally occurring peptide,often because the mimetic has a basic structure that mimics the basicstructure of the naturally occurring peptide and/or has the salientbiological properties of the naturally occurring peptide. Mimetics caninclude, but are not limited to: peptides that have substantialmodifications from the prototype such as no side chain similarity withthe naturally occurring peptide (such modifications, for example, maydecrease its susceptibility to degradation); anti-idiotypic and/orcatalytic antibodies, or fragments thereof; non-proteinaceous portionsof an isolated protein (e.g., carbohydrate structures); or synthetic ornatural organic molecules, including nucleic acids and drugs identifiedthrough combinatorial chemistry, for example. Such mimetics can bedesigned, selected and/or otherwise identified using a variety ofmethods known in the art. Various methods of drug design, useful todesign mimetics or other therapeutic compounds useful in the presentinvention are disclosed in Maulik et al., 1997, Molecular Biotechnology:Therapeutic Applications and Strategies, Wiley-Liss, Inc., which isincorporated herein by reference in its entirety.

[0083] A POMC mimetic (e.g., an MSH mimetic) can be obtained, forexample, from molecular diversity strategies (a combination of relatedstrategies allowing the rapid construction of large, chemically diversemolecule libraries), libraries of natural or synthetic compounds, inparticular from chemical or combinatorial libraries (i.e., libraries ofcompounds that differ in sequence or size but that have the similarbuilding blocks) or by rational, directed or random drug design. See forexample, Maulik et al., supra.

[0084] In a molecular diversity strategy, large compound libraries aresynthesized, for example, from peptides, oligonucleotides, carbohydratesand/or synthetic organic molecules, using biological, enzymatic and/orchemical approaches. The critical parameters in developing a moleculardiversity strategy include subunit diversity, molecular size, andlibrary diversity. The general goal of screening such libraries is toutilize sequential application of combinatorial selection to obtainhigh-affinity ligands for a desired target, and then to optimize thelead molecules by either random or directed design strategies. Methodsof molecular diversity are described in detail in Maulik, et al., ibid.

[0085] Maulik et al. also disclose, for example, methods of directeddesign, in which the user directs the process of creating novelmolecules from a fragment library of appropriately selected fragments;random design, in which the user uses a genetic or other algorithm torandomly mutate fragments and their combinations while simultaneouslyapplying a selection criterion to evaluate the fitness of candidateligands; and a grid-based approach in which the user calculates theinteraction energy between three dimensional receptor structures andsmall fragment probes, followed by linking together of favorable probesites.

[0086] Preferred POMC analogs (homologues or mimetics) for use orevaluation in the method of the present invention include POMC analogs(agonists or antagonists) of the melanocortins. Particularly preferredPOMC analogs for evaluation in the method of the present inventioninclude analogs of MSH proteins (peptides). Numerous analogs (homologuesand mimetics) of Pomc peptides, and particularly, of melanocortins, havebeen previously described in the art, and all are intended to beencompassed for use in the method of the present invention. For example,such analogs are disclosed in Hadley et al., 1986, “α-Melanotropinanalogs for Biomedical Applications”, Neural and Endocrine Peptides andReceptors, T. W. Moody, ed., Plenum Publ. Corp., NY, pp. 45-56; U.S.Pat. No. 4,649,191 to Hruby, U.S. Pat. No. 4,918,055 to Hruby et al.,U.S. Pat. No. 5,674,839 to Hruby et al., U.S. Pat. No. 5,683,981 toHadley et al., U.S. Pat. No. 5,714,576 to Hruby et al., and U.S. Pat.No. 5,731,408 to Hruby et al., each of which is incorporated herein byreference in its entirety, particularly with regard to the structures ofanalogs of melanocortins and especially, MSH analogs, disclosed therein,as well as to the methods of producing such analogs. An MSH agonistanalog suitable for use in the method of the present invention is[Ac-Cys⁴, D-Phe⁷, Cys¹⁰] α-MSH), although it will be apparent to thoseof skill in the art that the present invention is not limited to thisparticular MSH analog.

[0087] Some MSH analogs known in the art include, but are not limitedto, the following analogs:

[0088] (a) cyclic and linear α-MSH fragment analogs of the core sequenceof α-MSH, Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰ (positions 4-10 of SEQ IDNO:2), having modifications including but not limited to: (1)replacement of Met⁴ with Nle; (2) replacement of L-Phe⁷ with D-Phe⁷; (3)cyclization between positions 4 and 10; and/or (4) presence of Lys¹¹ inanalog at position 10 (See U.S. Pat. Nos. 5,674,839 and 5,714,576 toHruby et al., supra);

[0089] (b) linear and cyclic analogs of α-MSH having the generalformula:

Ac-[Nle⁴, X_(aa) ⁵, His⁶, X_(aa) ⁷, Arg⁷, Trp⁹, X_(aa) ¹⁰]-NH₂  (SEQ IDNO:3)

[0090] wherein X_(aa) ⁵ is either Glu or Asp, X_(aa) ⁷ is Phe or D-Pheand X_(aa) ¹⁰ is a dibasic amino acid, lysine, ornithine,2,4,-diaminobutyric acid, or 2,3 diaminopropionic acid (Dpr); and,

[0091] wherein cyclization is between positions 4 and 10 (See U.S. Pat.Nos. 5,674,839 and 5,714,576 to Hruby et al., supra);

[0092] (c) cyclic analogs of α-MSH using pseudoisosteric replacement ofMet⁴ and Gly¹⁰ with Cys amino acids Ac-[Cys⁴, Cys¹⁰]α-MSH₁₋₁₃NH₂ (SeeU.S. Pat. Nos. 5,674,839 and 5,714,576 to Hruby et al., supra);

[0093] (d) linear analogs of the formula: R₁-W-X-Y-Z-R₂ (See U.S. Pat.No. 4,918,055 to Hruby et al., supra); wherein

[0094] R₁ is selected from the group consisting of Ac-Gly-, Ac-Met-Glu-,Ac-Nle-Glu- and Ac-Tyr-Glu-;

[0095] W is selected from the group consisting of -His- and -D-His-;

[0096] X is selected from the group consisting of -Phe-, -D-Phe-, -Tyr,-D-Tyr-, (-pNO₂)D-Phe⁷-;

[0097] Y is selected from the group consisting of -Arg- and -D-Arg-;

[0098] Z is selected from the group consisting of -Trp- and -D-Trp-;and,

[0099] R₂ is selected from the group consisting of —NH₂, -Gly-NH₂, and-Gly-Lys-NH₂;

[0100] (e) linear α-MSH analogs having the formula:

[0101] Ac-Ser-Tyr-Ser-M-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQID NO:4), wherein M is selected from the group consisting of Met, Nle,and Cys (See U.S. Pat. No. 4,918,055 to Hruby et al., supra);

[0102] (f) linear α-MSH analogs selected from the group consisting of:

[0103] [Nle⁴, D-Phe⁷]-α-MSH;

[0104] [Nle⁴, D-Phe⁷]-α-MSH_(4-10;)

[0105] [Nle⁴, D-Phe⁷]-α-MSH_(4-11,)

[0106] [Nle⁴, D-Phe⁷, D-Trp⁹]-α-MSH₄₋₁₁; and,

[0107] [Nle⁴, D-Phe⁷]-α-MSH₄₋₉ (See U.S. Pat. No. 4,918,055 to Hruby etal., supra); and,

[0108] (g) cyclic bridged analogs of A-MSH having the general structure(See U.S. Pat. No. 5,683,981 to Hadley et al., supra)

[0109] wherein AA⁵ may be either a L- or D-amino acid having an omegaamino or carboxyl group in the side chain, e.g., α,γ-diaminopropionicacid, α,γ-diaminobutyric acid, Orn, Lys, α,β-aminoadipic acid,α-aminopimelic acid, or higher homologs, Glu or Asp;

[0110] wherein AA¹⁰ may be diaminopropionic acid, α,γ-diaminobutyricacid, Orn, Lys, α,β-aminoadipic acid, α-aminopimelic acid, or higherhomologs, Glu or Asp;

[0111] wherein R₁ is the designation α-MSH₁₋₁₃NH₂, α-MSH₁₋₁₂NH₂,α-MSH₁₋₁₁NH₂, α-MSH₄₋₁₃NH₂, or α-MSH₄₋₁₀NH₂;

[0112] wherein AA¹¹ may be L-or D-amino acid having an omega-amino orcarboxyl group in the side chain, e.g., α,β-diaminopropionic acid;α,γ-diaminobutyric acid, Orn, Lys, α-aminoadipic acid, α-aminopimelicacid, or higher homologs, Glu or Asp;

[0113] wherein R₂ is the designation α-MSH₁₋₁₃NH₂, α-MSH₁₋₁₂NH₂,α-MSH₁₋₁₁NH₂, α-MSH₄₋₁₃NH₂, or α-MSH₄₋₁₀NH₂; and,

[0114] wherein Xxx may be from 1 to 5 a-amino acid residues each ofwhich may be of L- or D-configuration, or a linear or branched chainspacer.

[0115] MSH analogs which may be particularly useful as α-MSH antagonists(See U.S. Pat. No. 4,649,191 to Hruby et al., supra) include, but arenot limited to:

[0116] (a) cyclic analogs having the general formula (See U.S. Pat. No.5,731,408 to Hadley et al., supra):

[0117] (b) cyclic analogs having the general formula (See U.S. Pat. No.4,649,191 to Hruby et al., supra):

[0118] wherein R¹ is a substituted or unsubstituted aromatic radical;

[0119] R² is hydrogen or a methyl group;

[0120] R³ is a carboxylate, carboxamide, hydroxymethyl, or aldehydegroup;

[0121] R⁴ is glutamic acid, alanine, -amino butyric acid, valine,leucine or isoleucine;

[0122] R⁵ is histidine, glutamic acid, alanine, valine, leucine orisoleucine;

[0123] R⁶ and R⁷, which may be the same or different, are hydrogen,methyl or lower alkyl having one to five carbon atoms;

[0124] R⁸ and R⁹, which may be the same or different, are hydrogen,methyl or lower alkyl having one to five carbon atoms;

[0125] X and Y are sulfur, methylene, SO or SO₂;

[0126] Z is —NH₂,

[0127] and,

[0128] n is an integer greater than or equal to 2;

[0129] (2)

[0130] wherein R¹ is phenyl, indole, p-hydroxyphenyl, p-aminophenyl,imidazole, 1-naphthyl adamantyl or alkylphenyl, 2-naphthyl;

[0131] R² is hydrogen or a methyl group;

[0132] R³ is a carboxylate, carboxamide, hydroxymethyl, or aldehydegroup;

[0133] X and Y are sulfur, methylene, SO or SO₂; Z is —NH₂,

[0134] and,

[0135] n is an integer greater than or equal to 2; and wherein thecyclized portion of the compound is conformationally restricted in amanner which is compatible with the reactivity of the compound withreceptors of the central nervous system.

[0136] In one aspect of the present invention, a compound useful as anMSH agonist or antagonist is an antibody, or an antigen binding fragmentthereof. In one aspect, the antibody selectively binds to an MSHreceptor in a manner such that the receptor is activated and therefore,such an antibody is considered to be an MSH agonist. In anotherembodiment, the antibody selectively binds to an MSH receptor in amanner that prevents binding of the receptor by a natural or syntheticagonist ligand, or that otherwise inhibits the activation of thereceptor, such antibody being an MSH antagonist. Alternatively, an MSHantagonist antibody can bind to an MSH agonist such that the MSH agonistis blocked or inhibited from binding to its receptor, and/or isotherwise eliminated from circulation. As used herein, the phrase“selectively binds” refers to the specific binding of one protein toanother (e.g., an antibody, fragment thereof, or binding partner to anantigen), wherein the level of binding, as measured by any standardassay (e.g., an immunoassay), is statistically significantly higher thanthe background control for the assay. For example, when performing animmunoassay, controls typically include a reaction well/tube thatcontain antibody or antigen binding fragment alone (i.e., in the absenceof antigen), wherein an amount of reactivity (e.g., non-specific bindingto the well) by the antibody or antigen binding fragment thereof in theabsence of the antigen is considered to be background. Binding can bemeasured using a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.

[0137] Isolated antibodies of the present invention can include serumcontaining such antibodies, or antibodies that have been purified tovarying degrees. Whole antibodies of the present invention can bepolyclonal or monoclonal, monovalent or bivalent. Alternatively,functional equivalents of whole antibodies, such as antigen bindingfragments in which one or more antibody domains are truncated or absent(e.g., Fv, Fab, Fab′, or F(ab)₂ fragments), as well asgenetically-engineered antibodies or antigen binding fragments thereof,including single chain antibodies or antibodies that can bind to morethan one epitope (e.g., bi-specific antibodies), or antibodies that canbind to one or more different antigens (e.g., bi- or multi-specificantibodies), may also be employed in the invention.

[0138] Genetically engineered antibodies of the invention include thoseproduced by standard recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Particular examples include, chimeric antibodies,where the V_(H) and/or V_(L) domains of the antibody come from adifferent source to the remainder of the antibody, and CDR graftedantibodies (and antigen binding fragments thereof), in which at leastone CDR sequence and optionally at least one variable region frameworkamino acid is (are) derived from one source and the remaining portionsof the variable and the constant regions (as appropriate) are derivedfrom a different source.

[0139] Construction of chimeric and CDR-grafted antibodies aredescribed, for example, in European Patent Applications: EP-A 0194276,EP-A 0239400, EP-A 0451216 and EP-A 0460617.

[0140] Generally, in the production of an antibody, a suitableexperimental animal, such as, for example, but not limited to, a rabbit,a sheep, a hamster, a guinea pig, a mouse, a rat, or a chicken, isexposed to an antigen against which an antibody is desired. Typically,an animal is immunized with an effective amount of antigen that isinjected into the animal. An effective amount of antigen refers to anamount needed to induce antibody production by the animal. The animal'simmune system is then allowed to respond over a pre-determined period oftime. The immunization process can be repeated until the immune systemis found to be producing antibodies to the antigen. In order to obtainpolyclonal antibodies specific for the antigen, serum is collected fromthe animal that contains the desired antibodies (or in the case of achicken, antibody can be collected from the eggs). Such serum is usefulas a reagent. Polyclonal antibodies can be further purified from theserum (or eggs) by, for example, treating the serum with ammoniumsulfate.

[0141] Monoclonal antibodies may be produced according to themethodology of Kohler and Milstein (Nature 256:495-497,1975). Forexample, B lymphocytes are recovered from the spleen (or any suitabletissue) of an immunized animal and then fused with myeloma cells toobtain a population of hybridoma cells capable of continual growth insuitable culture medium. Hybridomas producing the desired antibody areselected by testing the ability of the antibody produced by thehybridoma to bind to the desired antigen.

[0142] Alternative methods, employing, for example, phage displaytechnology (see for example U.S. Pat. No. 5,969,108, U.S. Pat. No.5,565,332, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657) or theselected lymphocyte antibody method of U.S. Pat. No. 5,627,052 may alsobe used for the production of antibodies and/or antigen fragments of theinvention, as will be readily apparent to the skilled individual.

[0143] The invention also extends to non-antibody polypeptides,sometimes referred to as binding partners, that have been designed tobind specifically to, and either activate or inhibit as appropriate, MSHor an MSH receptor according to the present invention. Examples of thedesign of such polypeptides, which possess a prescribed ligandspecificity are given in Beste et al. (Proc. Natl. Acad. Sci.96:1898-1903, 1999), incorporated herein by reference in its entirety.

[0144] In another aspect of the present invention, a compound useful asan MSH antagonist is a soluble MSH receptor (e.g., an isolatedextracellular portion of the receptor, or a portion or fragment thereofthat binds to an MSH agonist). As disclosed by U.S. Pat. Nos. 5,908,609and 5,932,779, the cloning and characterization of each receptor hasbeen described: MC1-R and MC2-R (Mountjoy, 1992, Science, 257:1248-1251;Chhajlani & Wikberg, 1992, FEBS Lett., 309:417-420); MC3-R(Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci. USA, 90:8856-8860;Gantz et al., 1993, J. Biol. Chem., 268:8246-8250); MC4-R (Gantz et al.,1993, J. Biol. Chem., 268:15174-15179; Mountjoy et al., 1994, Mol.Endo., 8:1298-1308); and MC5-R (Chhajlani et al., 1993, Biochem.Biophys. Res. Commun., 195:866-873; Gantz et al., 1994, Biochem.Biophys. Res. Commun., 200:1214-1220), each of which is incorporated byreference herein in its entirety. Thus, each of the foregoing sequencescan be utilized to engineer a cell or cell line that expresses one ofthe melanocortin receptors, or a soluble receptor portion for use in themethods described herein.

[0145] According to the present invention, an isolated or biologicallypure protein, including peptides and analogs thereof, is a protein thathas been removed from its natural milieu. As such, “isolated” and“biologically pure” do not necessarily reflect the extent to which theprotein has been purified. An isolated protein of the present inventioncan be obtained from its natural source, can be produced usingrecombinant DNA technology or can be produced by chemical synthesis.Such methods are described in detail below. It is to be noted that theterm “a” or “an” entity refers to one or more of that entity; forexample, a compound refers to one or more compounds or at least onecompound. As such, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, and “having” can be usedinterchangeably. Furthermore, a compound “selected from the groupconsisting of” refers to one or more of the compounds in the list thatfollows, including mixtures (i.e., combinations) of two or more of thecompounds.

[0146] The compounds useful for induction of insulin-resistance (i.e.,compounds having MSH biological activity) and/or for evaluation for theability to reduce insulin resistance in the method of the presentinvention may be produced by any method suitable for the production ofpeptides and/or non-peptide mimetics, and particularly, for Pomcpeptides or non-peptide mimetics. For example, such methods include wellknown chemical procedures, such as solution or solid-phase peptidesynthesis, or semi-synthesis in solution beginning with proteinfragments coupled through conventional solution methods. Such methodsare well known in the art and may be found in general texts and articlesin the area such as: Merrifield, 1997, Methods Enzymol. 289:3-13; Wadeet al., 1993, Australas Biotechnol. 3(6):332-336; Wong et al., 1991,Experientia 47(11-12):1123-1129; Carey et al., 1991, Ciba Found Symp.158:187-203; Plaue et al., 1990, Biologicals 18(3): 147-157; Bodanszky,1985, Int. J Pept. Protein Res. 25(5):449-474; or H. Dugas and C.Penney, BIOORGANIC CHEMISTRY, (1981) at pages 54-92, all of which areincorporated herein by reference in their entirety. For example,peptides may be synthesized by solid-phase methodology utilizing acommercially available peptide synthesizer and synthesis cycles suppliedby the manufacturer. One skilled in the art recognizes that the solidphase synthesis could also be accomplished using the FMOC strategy and aTFA/scavenger cleavage mixture. Methods for synthesizing MSH analogs,for example, are described in detail in U.S. Pat. No. 4,649,191 toHruby, supra, U.S. Pat. No. 4,918,055 to Hruby et al., supra, U.S. Pat.No. 5,674,839 to Hruby et al., supra, U.S. Pat. No. 5,683,981 to Hadleyet al., supra, U.S. Pat. No. 5,714,576 to Hruby et al., supra, and U.S.Pat. No. 5,731,408 to Hruby et al., supra, all of which are incorporatedherein by reference in their entirety.

[0147] If larger quantities of a Pomc peptide are desired, the peptide(or peptide analog thereof) can be produced using recombinant DNAtechnology, although for proteins of this small size (i.e., peptides),peptide synthesis is generally more preferred. A peptide can be producedrecombinantly by culturing a cell capable of expressing the peptide(i.e., by expressing a recombinant nucleic acid molecule encoding thepeptide) under conditions effective to produce the peptide, andrecovering the peptide. Such techniques are well known in the art andare described, for example, in Sambrook et al. supra.

[0148] In the practice of the method of the present invention, it isuseful, although not essential, to prepare formulations comprising anamount of at least one compound having MSH biological activity or atleast one regulatory compound to be evaluated or to be administered fora therapeutic purpose, either alone or in combination with apharmaceutically acceptable salt and/or complexed with another suitablecarrier (described below). Such formulations can be formulated for anyroute of administration, including, but not limited to, parenteraladministration and transdermal administration. For example, formulationsto be evaluated can be formulated in an excipient that the animal to betreated can tolerate. Examples of such excipients include water, saline,phosphate buffered solutions, Ringer's solution, dextrose solution,Hank's solution, polyethylene glycol-containing physiologically balancedsalt solutions, and other aqueous physiologically balanced saltsolutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyloleate, or triglycerides may also be used.

[0149] The formulations comprising one or more desired compoundstypically contain from about 0.1% to 90% by weight of the activecompound, preferably in a soluble form, and more generally from about0.1% to 1.0%.

[0150] In one embodiment of the present invention, a pharmaceuticallyacceptable carrier can include additional compounds that increase thehalf-life of a formulation in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled release vehicles,biodegradable implants, liposomes, bacteria, viruses, other cells, oils,esters, and glycols.

[0151] In one embodiment of the present invention, a formulation caninclude a controlled release composition that is capable of slowlyreleasing the formulation into an animal. As used herein, a controlledrelease composition comprises a regulatory compound to be evaluated asdescribed herein in a controlled release vehicle. Suitable controlledrelease vehicles include, but are not limited to, biocompatiblepolymers, polymeric matrices, capsules, microcapsules, microparticles,bolus preparations, osmotic pumps, diffusion devices, liposomes,lipospheres, and transdermal delivery systems. Other controlled releasecompositions of the present invention include liquids that, uponadministration to an animal, form a solid or a gel in situ. Preferredcontrolled release compositions are biodegradable (i.e., bioerodible).

[0152] According to the present invention, an effective administrationprotocol (i.e., administering a regulatory compound or a formulationcomprising such a compound in an effective manner) comprises suitabledose parameters and modes of administration that are not toxic to theanimal, and which would reasonably be expected to provide a measurablechange in the insulin resistance (or sensitivity) in the animal whenadministered one or more times over a suitable time period. It is wellwithin the ability of one of skill in the art to establish a suitabledose and administration protocol for evaluating the ability of acompound to regulate insulin resistance in a genetically modifiednon-human animal of the present invention. Effective dose parameters canbe determined using methods standard in the art for a particular animaland condition. Such methods include, for example, determination ofsurvival rates, side effects (i.e., toxicity) and other health factorsassociated with the administration of the compound.

[0153] Modes of administration of a compound or formulation of thepresent invention include any method of administration which results indelivery of the composition to the animal and particularly, tomelanocortin receptors in tissues of the animal. Such modes ofadministration can include, but are not limited to, oral, nasal,topical, transdermal, rectal, and parenteral routes. Parenteral routescan include, but are not limited to subcutaneous, intradermal,intravenous, intraperitoneal and intramuscular routes. In oneembodiment, the route of administration is by topical or transdermaladministration, such as by a lotion, cream, a patch, an injection, animplanted device (e.g., similar to Norplant), or other controlledrelease carrier.

[0154] In the embodiment where the compound or formulation is to bedelivered to a patient in the form of a nucleic acid molecule encoding apeptide compound to be evaluated, the nucleic acid molecules can bedelivered to a patient by a variety of methods including, but notlimited to, (a) administering a naked (i.e., not packaged in a viralcoat or cellular membrane) nucleic acid molecule (e.g., as naked DNA orRNA molecules, such as is taught, for example in Wolff et al., 1990,Science 247, 1465-1468); (b) administering a nucleic acid moleculepackaged as a recombinant virus, in a liposome delivery vehicle, or in arecombinant cell (i.e., the nucleic acid molecule is delivered by aviral or cellular vehicle); or (c) administering a recombinant nucleicacid molecule encapsulated within a liposome delivery vehicle.

[0155] The final step in the method of identifying a compound accordingto the present invention is a step of selecting a compound from step (b)that decreases the insulin resistance in said non-human animal ascompared to in the absence of said compound of (b). Since the presentinventors have discovered that the administration of MSH is capable ofinducing resistance to insulin in the genetically modified POMC nullmutant of the invention, one can now select compounds that have theability to prevent or reverse this effect. Insulin resistance (orsensitivity) can be measured by any suitable method known in the art,including, but not limited to, measurement of blood glucose during aninsulin tolerance test (e.g., see Example 2), or any other suitablemethod as previously described herein. Other methods for measuring orevaluating the biological processes associated with insulin resistanceinclude, but are not limited to, assaying the mutant strains forpancreatic β cell mass through morphometric analysis and for β cellfunction through analysis of glucose-stimulated insulin release in vivo;assaying the insulin receptor in muscle, adipose tissue, and liver frommutant strains with respect to its number on tissue membranes, itsbinding affinity, its tyrosine kinase activity, and its phosphorylationstate, as well as analyzing the insulin receptor signaling pathwaythrough determining phosphorylation of IRS1 and IRS-2, and activation ofPEPCK, PI 3-kinase, MAP kinase, as well as expression of TNF-α, PTP-1Band PPARγ. Regulatory compounds that decrease or prevent the insulinresistance that is induced by administration of the compound having MSHbiological activity (i.e., as compared to in the absence of theregulatory compound), are selected as regulatory agents with potentialfor reducing insulin resistance in an animal that has insulinresistance, such as in an obese animal and/or an animal that has type IIdiabetes. Additional controls that may be used in the method of thepresent invention include heterozygous Pomc mutants and wild-type mice,which can be used to confirm that the action of the regulatory compoundis related to the effects of the pomc locus and particularly, MSH.

[0156] Another embodiment of the present invention relates to a methodto reduce insulin resistance in a mammal, or to prevent or treatdiabetes in a mammal, such methods comprising administering to a mammalthat has insulin resistance a therapeutic composition comprising anantagonist of melanocortin stimulating hormone (MSH) biologicalactivity, wherein the antagonist decreases insulin resistance in saidmammal. Insulin resistance, and methods of measuring insulin resistance,have been described previously herein. According to the presentinvention, the phrase, “to reduce insulin resistance” in a patientrefers to any detectable reduction of insulin resistance as compared toa previous level of insulin resistance or to a standard control levelestablished for the patient or for the general patient based on species,age, race, or another factor(s). To “treat” a disorder, such as adisorder associated with insulin resistance (e.g., type II diabetes)refers to reducing or ameliorating the disorder in a patient thatsuffers from the disorder, and to “prevent” a disorder refers to haltingthe disorder in a patient that is at risk of suffering from the disorderbefore the disorder becomes overt. Preferably, the disorder, or thepotential for developing the disorder, is reduced, optimally, to anextent that the patient no longer suffers from or does not develop thedisorder (e.g., excessive accumulation of fat stores in adipose tissue),or the discomfort and/or altered functions and detrimental conditionsassociated with such disorder.

[0157] This method of the invention comprises the use of an antagonistof melanocortin stimulating hormone (MSH) biological activity.Antagonists of MSH biological activity have been described in detailpreviously herein, and include, but are not limited to, a protein, apeptide, a nucleic acid, an antibody or antigen-binding fragmentthereof, a carbohydrate-based compound, a lipid-based compound, anatural organic compound, a synthetically derived organic compound, asoluble MSH receptor, or other compound (e.g., any product of drugdesign) that selectively binds to and blocks access to the receptor by anatural or synthetic agonist ligand (e.g., by binding to either thereceptor or a natural or synthetic agonist of the receptor) or reducesor inhibits the activity of an MSH receptor; or a product of drug designthat blocks the receptor or alters the biological activity of thereceptor. Preferably, a therapeutic composition comprising a compoundthat is an antagonist of MSH biological activity, alone or incombination with one or more additional compounds useful for reducinginsulin resistance or treating diabetes, if the patient has diabetes, isformulated to be administered in a manner which extends the time thecomposition remains in the bloodstream of an animal. As such, atherapeutic composition of the present invention typically includes apharmaceutically acceptable carrier, and preferably, one which iscapable of delivering the composition of the present invention tomelanocortin receptors in the animal, and in some cases, is capable ofprolonging the action of the composition in the bloodstream of theanimal.

[0158] For example, therapeutic compositions (i.e., formulations) of thepresent invention can be formulated in an excipient that the animal tobe treated can tolerate. Examples of such excipients include water,saline, phosphate buffered solutions, Ringer's solution, dextrosesolution, Hank's solution, polyethylene glycol-containingphysiologically balanced salt solutions, and other aqueous,physiologically balanced, salt solutions. Nonaqueous vehicles, such asfixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.Other useful formulations include suspensions containing viscosityenhancing agents, such as sodium carboxymethylcellulose, sorbitol, ordextran. Excipients can also contain minor amounts of additives, such assubstances that enhance isotonicity and chemical stability or buffers.Examples of buffers include phosphate buffer, bicarbonate buffer andTris buffer, while examples of preservatives include thimerosal, m- oro-cresol, formalin and benzyl alcohol. Standard formulations can eitherbe liquid injectables or solids which can be taken up in a suitableliquid as a suspension or solution for injection. Thus, in a non-liquidformulation, the excipient can comprise dextrose, human serum albumin,preservatives, etc., to which sterile water or saline can be added priorto administration.

[0159] Preferred slow-release compositions have been previouslydescribed herein.

[0160] The compositions comprising one or more desired compoundstypically contain from about 0.1% to 90% by weight of the activecompound, preferably in a soluble form, and more preferably, from about0.1% to about 50%, and more preferably from about 0.1% to about 25%, andeven more preferably, from about 0.1% to about 10%, and even morepreferably, from about 0.1% to 1.0%.

[0161] In one aspect of the present method, a transdermal patch can beused to deliver the therapeutic composition of the present invention.Such a patch can include additional compounds for enhancing the delivery(i.e., transfer) of components across the epidermal surface of the skinand into the peripheral circulation (e.g., DMSO).

[0162] A preferred controlled release composition of the presentinvention is capable of releasing a formulation of the present inventioninto the blood of an animal at a constant rate sufficient to maintaintherapeutic levels of the formulation to decrease insulin resistanceover a period of time ranging from days to months based on toxicityparameters. A controlled release formulation of the present invention iscapable of effecting control over insulin resistance for preferably atleast about 6 hours, more preferably at least about 24 hours, and evenmore preferably for at least about 7 days.

[0163] According to the present invention, an effective administrationprotocol (i.e., administering a compound that is an antagonist of MSHbiological activity or a therapeutic composition comprising such acompound in an effective manner) comprises suitable dose parameters andmodes of administration that result in regulation of insulin resistancein the animal when administered one or more times over a suitable timeperiod. In one embodiment, an effective administration protocol resultsin a measurable reduction of insulin resistance in an animal within atleast about 2 weeks after the first administration of the MSH antagonistcompound, and more preferably, within at least one week, and morepreferably, within at least 3 days, and even more preferably, within atleast 24 hours of the first administration of an MSH antagonistcompound.

[0164] Effective dose parameters can be determined using methodsstandard in the art for a particular animal and condition. Such methodsinclude, for example, determination of survival rates, side effects(i.e., toxicity) and other health factors associated with, or inaddition to the regulation of insulin resistance in the animal. Inparticular, the effectiveness of dose parameters of a therapeuticcomposition of the present invention when used to control insulinresistance can be determined by assessing response rates. Such responserates can refer to the percentage of treated patients in a population ofpatients that respond with a detectable reduction in insulin resistance,or to the insulin response of the individual patient, as compared to aprevious measurement of insulin response in the patient prior to thestart of treatment, to a level which is considered by those of skill inthe art to be sufficient to address the needs of the particular patientand/or not present health risks to the patient. Response can bedetermined by, for example, measuring insulin resistance/sensitivityover time and/or measuring changes in other indicators of insulinfunction, for example, β cell mass, insulin receptor numbers and/orfunction, etc.

[0165] Modes of administration of a therapeutic composition of thepresent invention include any method of administration which results indelivery of the composition to the peripheral or central circulation ofthe animal. Such modes of administration can include, but are notlimited to, oral, nasal, topical, transdermal, rectal, and parenteralroutes, as well as direct injection into a tissue and delivery by acatheter. Parenteral routes can include, but are not limited tosubcutaneous, intradermal, intravenous, intraperitoneal andintramuscular routes. In one embodiment, the route of administration isby topical or transdermal administration, such as by a lotion, cream, apatch, an injection, an implanted device (e.g., similar to Norplant), orother controlled release carrier. Preferred routes of administrationinclude transdermal delivery and delivery via an implanted device orother controlled release carrier. Particularly preferred routes ofadministration include any route which directly delivers the compositionto the systemic circulation (e.g., by injection), including anyparenteral route. It is noted that one of skill in the art will be ableto use the guidance provided herein regarding route of administration,pharmaceutical carriers or excipients, and dosage, to select anadministration protocol which delivers the composition of the presentinvention to the melanocortin receptors to effect insulin resistance inthe animal, as opposed to, for example, merely delivering thecomposition to the dermal tissue as has been previously described forMSH for the use in the treatment of dermal conditions (e.g., vitaligo ordermatitis). Although topical and transdermal delivery of MSH andanalogs thereof has been described prior to the present invention (e.g.,U.S. Pat. No. 4,874,744 to Nordlund or U.S. Pat. No. 4,649,191 to Hrubyet al.), such methods were directed to the treatment of conditions atthe dermis, and therefore these methods taught dosage protocols,carriers and administration methods which were suitable for deliveringMSH to the dermis for action at the skin, but failed to describe doses,carriers and/or administration methods that are suitable for delivery ofMSH antagonists for the prevention and/or treatment of insulinresistance and diabetes associated therewith. For example, these methodstypically suggested concentration ranges for MSH (e.g., 10⁻¹⁰, 10⁻¹¹)which are well below the level which would be expected to provide asignificant effect in the method of the present invention.

[0166] In accordance with the present invention, a suitable or effectivesingle dose size is a dose that is capable of causing a measurablechange in insulin resistance/sensitivity (e.g., a decrease in insulinsensitivity) of a patient when administered one or more times over asuitable time period. A suitable or effective single dose size can alsobe a dose that is capable of causing a measurable change in insulinresistance in a patient as compared to the measure of insulin resistanceestablished prior to initiation of the treatment, when administered oneor more times over a suitable time period. Doses can vary depending uponthe condition of the patient being treated, including the severity ofthe insulin resistance, whether the patient suffers from overt diabetesor not, and/or any other related or non-related health factorsexperienced by a particular patient. Typically, the method of thepresent invention comprises administering a compound having MSHantagonist activity in a dose between about 0.1 μg and about 100 mg perkilogram body weight of the patient, and preferably, between about 0.1μg and about 10 mg per kilogram body weight of the patient, and morepreferably, between about 0.1 μg and about 1 μg per kilogram body weightof the patient, and even more preferably, between about 1 μg and about10 mg per kilogram body weight of the patient. A more preferred singledose is from about 40 μg to about 1 mg per kilogram body weight of thepatient. A typical daily dose for an adult human (i.e., a 75 kg human)is from about 1 milligram to about 100 milligrams. A preferredcirculating level of a compound to achieve in a patient regardless ofthe route of administration is from about 0.1 μg per kilogram bodyweight to about 10 μg per kilogram body weight, and more preferably,from about 0.1 μg per kilogram body weight to about 1 μg per kilogrambody weight of the patient. In practicing this method, the compound ortherapeutic composition containing the compound can be administered in asingle daily dose or in multiple doses per day. This treatment methodmay require administration over extended periods of time. The amount peradministered dose or the total amount administered will be determined bythe physician and will depend on such factors as the mass of thepatient, the age and general health of the patient and the tolerance ofthe patient to the compound.

[0167] In the methods of the present invention, a therapeutic compound,including agonists and antagonists of MSH, as well as compositionscomprising such compounds, can be administered to any organism, andparticularly, to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Preferred mammals to treatinclude humans. Preferably, mammals to treat using the method of thepresent invention have, or are at risk of developing, insulin resistanceand/or diabetes associated with insulin resistance (e.g., non-insulindependent diabetes mellitus). Methods and standards for identifying suchindividuals are well known in the art.

[0168] According to the present invention, the phrases “type IIdiabetes”, “type 2 diabetes”, “non-insulin dependent diabetes mellitus”and “NIDDM” refer to the same condition. Individuals with NIDDM aretypically overweight or obese at diagnosis and present with glycosuriawithout ketonuria, absent or mild polyuria and polydipsia, and little orno weight loss. Testing for Type 2 diabetes typically involves drawingblood samples and measuring the glucose (sugar) levels within the blood.During a random glucose test, a sample of blood can be obtained andtested at any time. Normal random glucose levels are 70-110 mg/dl.

[0169] According to the American Diabetes Association, a random glucoselevel of greater than 200 mg/dl is indicative of diabetes. During afasting glucose test, a sample of blood is obtained following a periodof not eating or drinking (except water) for at least 8 hours. It isusually drawn early in the morning, before breakfast. According to theAmerican Diabetes Association, a fasting blood glucose level of greaterthan 125 mg/dl on two occasions is indicative of diabetes. The fastingblood glucose test is the most common test in use for diagnosingdiabetes. During an oral glucose tolerance test, a fasting blood sugaris obtained initially. The person is then asked to drink a sweet sugarybeverage. Blood glucose levels are then obtained every 30 minutes forthe next 2 hours. A blood glucose level below 140 mg/dl at 2 hours isconsidered normal. A blood glucose level of greater than 200 mg/dl at 2hours is indicative of diabetes. A blood glucose level of 140-200 mg/dlat 2 hours indicates some impairment in glucose tolerance.

[0170] Various aspects of the present invention are illustrated in thefollowing examples, which are provided for the purposes of illustrationand are not intended to limit the scope of the present invention.

EXAMPLES Example 1

[0171] The following example describes the production of the POMC nullmutant mouse used in the present invention and demonstrates that Pomcpeptides are associated with the regulation of body weight through bothcentral and peripheral mechanisms.

[0172] To create a mutant mouse strain lacking all proopiomelanocortin(Pomc) derived peptides, the present inventors designed a targetingvector in which the entire third exon (Notake et al., 1983, FEBS Lett156:67-71; incorporated herein by reference in its entirety) is replacedby a neomycin resistance cassette. Briefly, EcoRI-digested 129/SvEvgenomic DNA was cloned into lambda FixII (Stratagene). The resultinglibrary was screened with a 0.3 kb PCR fragment from exon 3 of the mousePomcl sequence, and a clone carrying a 9.5 kb fragment containing themouse Pomcl locus was isolated. For the targeting vector the KnpI-PstIfragment containing the third exon was deleted. This removes all but thefirst 44 codons for amino acids after the translation start of thepre-pro-protein, or all but the first 18 codons for amino acids of thePOMC protein. Targeting vector (20 μg) was used to electroporate 10⁷ RW4ES cells (Genome Systems). ES cells which homologously integrated themutated allele were injected into C57BL/6 blastocysts as described(Hogan et al., “Manipulating the Mouse Embryo”, Cold Spring HarborLaboratory Press, 1994). Chimeric mice were mated to 129/SvEvTacfemales. Heterozygous offspring were mated to generate homozygous mutantmice. Genotypes were analyzed by PCR and confirmed by Southern blotanalysis as described (Sambrook et al. ibid.).

[0173]FIG. 1A shows schematic diagrams and restriction maps of the mousePomc locus, the targeting vector, and the predicted structure of thePomc locus after homologous recombination. The 0.4 kb probe fragmenthybridizes to a 9.5 kb EcoRI fragment in the wildtype allele, and to a3.2 kb fragment in the mutant allele (see also FIG. 1B). Restrictionsites indicated are EcoRI (E), KpnI (K), and PstI (P). FIG. 1B showsSouthern blot analyses of tail DNAs from F₂ littermates. The probe usedwas the 0.4 kb PstI-EcoRI fragment (see FIG. 1A). FIG. 1C shows an RIAanalysis of serum ACTH levels in F₂ male littermates (measurements intriplicates, one mouse per genotype) (discussed in detail below).

[0174] The deleted POMC allele construct was introduced into embryonicstem (ES) cells by electroporation and from there into the mousegermline, generating strain Pomc^(tm2ute). When the mutation wasbackcrossed into the inbred 129/SvEv background, homozygous Pomc mutantswere born to heterozygous parents at one quarter (39 wildtype, 80heterozygotes, 10 mutants) of the frequency expected for a recessivemutation, indicating that concurrent lack of all of the embryonicderived Pomc peptides is compatible with survival throughout prenataldevelopment in only a fraction of the animals.

[0175] Female POMC null mice are fertile and carry heterozygous andwild-type pups to term; male POMC null mice are infertile. Whenheterozygous POMC males are mated to homozygous Pomc mutant females,homozygous mutant, but not heterozygous, offspring die within the firstfew hours after birth.

[0176] During the first postnatal month homozygous mutants aresuperficially indistinguishable from their wildtype littermates. In thesecond month, mice lacking Pomc peptides start to gain weight visibly,and by the third postnatal month their weights are about twice those oftheir wildtype littermates (FIG. 2A; weight measurements were taken frommale mice of each genotype; at 2 months n=4, P<0.0005; at 3 months n=3,P<0.005). The weight gain is accompanied by both a slight, butsignificant, increase in body length (FIG. 2B; measurements (snout toroot of tail) were taken from 3-4 months old female mice, 6 mice pergenotype (P<0.001)) and a large increase in serum leptin levels (FIG.2C). In this latter experiment, serum leptin levels were determined (induplicates) from blood samples collected retroorbitally from 6-8 monthsold, individual, male and female mice. Average weights were 30.9 g forwildtype mice, 31.7 g for heterozygotes, and 55.9 g for homozygotes.Interestingly, heterozygote mice show elevated levels of serum leptin,but do not display increased body weight. The elevated leptin levels inthe normal weight heterozygotes suggest a homeostatic balance betweenleptin levels and Pomc peptide levels: the decreased Pomc peptide levelsare compensated by increased leptin. The mechanism and significance ofsuch a relationship suggest a paracrine feed-back loop.

[0177] It was also noticed that the Pomc mutant mice raised on a highfat breeder chow gained weight faster than mice raised on standard chow.Wildtype and mutant females (3 per test group) were given unlimitedaccess either to standard or to breeder chow (4.5% and 9% fat,respectively). FIGS. 2D and 2E show weight change (2D) and food intake(2E) during one week. Food intake in the “high fat diet” groups wasmeasured in bulk for all three mice. FIG. 2D shows that the mutant micegained 3 grams more per week on a high fat diet versus a standard diet(3.8 g versus 0.8 g), while wildtype mice gained 0.2 g more on a highfat diet versus a standard diet (0.7 g versus 0.5 g). FIG. 2E shows thatthe food intake by Pomc mutants increased with high fat diet by 2.4 g(30.3 g versus 32.7 g), while the food intake by wildtype littermatesdecreased with high fat diet by 2.2 g (23.5 g versus 21.3 g). Undereither dietary condition mutant mice lacking POMC have an increased foodintake compared to wildtype littermates. These results suggest that POMCderived peptides mediate both food intake and bodily food deposit.Wildtype mice regulate their food intake according to the diet, i.e.,they decrease intake with a higher caloric supply, and they adjust theirmetabolism (food deposit versus burning) to keep their body weightconstant. In contrast, mice lacking POMC show a deficit in both of theseaspects with the result of increased body weight: they have an increasedfood uptake and they lack the ability to catabolize dietary fat.

[0178] Another visible difference between POMC null mutant mice and thewildtype mice is the yellowish pigmentation of mutant mice (data notshown), which is especially pronounced on the belly. MC1-R inmelanocytes is normally stimulated by α-MSH, resulting in synthesis ofeumelanin (black/brown) pigment (Burchill et al., 1986, J. Endocrinol.109:15-21). Antagonism of MC1-R by the agouti-signaling protein (ASP)overexpressed in A^(y) mice results in whole body yellow coat color (Luet al., 1994, Nature 371:799-802). A loss-of-function mutation in theMclr gene in the recessive yellow mouse (e/e) (Robbins et al., 1993,Cell 72:827-834) and in cattle (Joerg et al., 1996, Mamm. Genome7:317-318) causes yellow coat and red coat, respectively. The humanpatients with POMC null mutations have red hair as well (Krude et al.,ibid.). In the POMC null mice, the change in pigmentation is subtle, inthat the coat covering the sides and belly is more yellow than inwildtype littermates, and the tips of the hairs at the back have ayellowish tinge. These pigmentation differences in mutants become morepronounced during adulthood. The fact that in the mouse, lack of theligand (Pomc) does not result in a phenotype congruent with lack orantagonism of MC1-R, suggests the presence of other ligands for thismelanocortin receptor. Alternatively, this result could be explained ifthere is a ligand-independent constitutive activity of the receptor.

[0179] Next, the effect of a complete lack of ACTH on adrenal functionwas determined. Serum corticosterone levels (FIG. 3A) were determined byRIA from blood samples collected retroorbitally from 6-7 month old mice(n=7 for wildtypes, n=6 for heterozygotes, n=5 for mutants). Serumaldosterone levels (FIG. 3B) were determined in trunk blood samples from7-8 month old mice (n=1 for wildtypes, n=2 for heterozygotes, n=3 formutants). Plasma catecholamine levels (FIGS. 3C-3E) were determined intrunk blood samples from 7-8 month old mice (n=4 for wildtype mice, n=3for mutant mice).

[0180]FIG. 1C shows an RIA analysis of serum ACTH levels in F₂ malelittermates (measurements in triplicates, one mouse per genotype). Bloodwas collected retroorbitally and serum was analyzed by RIA following theprovider's instructions (ICN, corticosterone; IncStar, ACTH; Linco,Leptin). FIG. 1C shows that ACTH levels in the mutant animal were belowthe sensitivity of the assay, indicating that the coding region for allPomc peptides had been deleted.

[0181] Serum corticosterone and aldosterone levels were below detection(FIGS. 3A and 3B), despite considerable stressing of mice during bloodcollection, indicating an absolute necessity for POMC derived peptidesfor adrenal cortical function. Here again, heterozygotes show a genedosage effect, suggesting fine-tuned regulation by Pomc peptides. Whenplasma catecholamine basal levels were measured (FIGS. 3C-3E),epinephrine was significantly lower in Pomc mutants versus wildtype mice(FIG. 3C; p<0.006), while levels of norepinephrine were notsignificantly altered (FIG. 3D; p<0.27) and dopamine levels wereslightly increased in mutants compared to wildtypes (FIG. 3E; p<0.06).In cases of dysfunction of the adrenal medulla, other chromaffinetissues expressing catecholamines increase production to compensate;epinephrine, however, is almost exclusively produced by the adrenalmedulla. The significant decrease of epinephrine indicates a severedysfunction and/or lack of the adrenal medulla in POMC deficient mice.Finding adrenal glands proved to be impossible: mutant mice had nomacroscopically discernible adrenal glands. For histological analysis,tissues from the fat pad surrounding the kidney were collected andimmediately placed into formalin. Sectioning (5 μm thickness) andstaining were carried out by American Histolab, Inc., Gaithersburg, Md.Histological examination of the fat pad surrounding the kidney andpresumably containing adrenal tissue revealed areas of tissuereminiscent of rudimentary adrenal medulla or adrenal cortex (data notshown). However, immunohistochemical staining with antibodies againstkey enzymes in catecholamine synthesis (PNMT and TH) were negative (datanot shown).

[0182] The lack of a normal adrenal gland structure in POMC null micepoints to a critical role of POMC derived peptide(s) in adrenaldevelopment. POMC adrenocorticotropin (ACTH) of pituitary origin is theonly known ligand for the MC2-R in the adrenal gland. It is surprisingthat loss of ligand (ACTH) results in loss of the tissue expressing itsreceptor (MC2-R). Without being bound by theory, the present inventorsbelieve that it may be more likely that another POMC factor distinctfrom ACTH plays a role as trophic factor in adrenal gland development.Candidate peptides would be peptides derived from the N-terminalnon-γ-MSH region of POMC (N—POMC₁₋₂₈. N—POMC₂₋₅₉), which have beenimplicated in the physiological control of adrenal growth (Estivariz etal., 1982, Nature 297:419-422). This can be tested by reconstituting thePOMC null mice with candidate peptides. It may also be possible at thatpoint to determine whether the lack of adrenal medulla is a consequenceof the lack of Pomc peptides, or of adrenal cortical structure, or ofadrenal cortical factors (i.e., corticosterone).

[0183] The phenotype of obesity, adrenal insufficiency, and alteredpigmentation, makes the POMC null mouse a model for the human POMC nullsyndrome. In the human POMC deficient patients and in the mouse Pomcmutant, homozygotes are born within the normal range of weight and size.Development of obesity starts at 4 to 5 months in the reported cases inhumans (Krude et al., 1998, Nat. Genet. 19:155-157), and at 1 month inPOMC null mice. This time course of obesity is also similar to that seenin fat/fat mice, which lack carboxypeptidase E, a prohormone processingenzyme (Naggert et al., 1995, Nat. Genet. 10:135-142). A defect inprocessing of POMC could explain the obesity component of the fat/fatphenotype.

[0184] In the human POMC deficient patients, ACTH deficiency results inhypocortisolism and, if untreated, in death. In the POMC null mice, thepresent inventors were unable to detect corticosterone in serum, evenunder moderate stress conditions. In contrast to humans, mice thatdevelop with maternal but without endogenous corticosterone are viable.A similar observation has been made in mice lacking corticotropinreleasing factor, CRH, which develop normally despite very low levels ofcorticosterone (Muglia et al., 1995, Nature 373:427-432). As inoffspring from CRH null females, homozygous offspring from POMC nullmutants die within the first hours after birth. This is probably due todefective lung maturation with the lack of corticosterone, as has beendemonstrated for the CRH null mutants.

[0185] Corticosteroids are known to increase food intake (Tempel et al.,1994, J. Neuroendocrinol. 6:479-501) and to decrease energy expenditure(Strack et al., 1995, Am. J. Physiol. 268:R1209-1206). POMC null micehave no detectable corticosterone, yet they are obese. This is so farthe only situation where obesity occurs in the absence ofcorticosterone. In all other forms of murine obesity, corticosterone isat normal or elevated levels. In fact, the excessive obesity inleptin-deficient mice is largely due to the hypercortisolism in thismouse and adrenalectomy blocks the development of excessive obesity inlep^(ob)/lep^(ob) mice (Solomon et al., 1973, Endocrinology 93:510-512and Tokuyama et al., 1989, Am. J. Physiol. 257:E139-144).

[0186] Lack of ligands for the melanocortin receptors in POMC-deficientmice replicate fully or partly the effects seen in mice lacking thereceptors MC3-R, MC4-R or MC1-R, respectively. In a preliminaryanalysis, POMC-deficient mice also replicate the defective waterrepulsion and thermoregulation seen in mutant mice lacking MC5-R (datanot shown). The present results provide a strong indication that Pomcpeptides are the physiological ligands for at least some MC5-R mediatedfunctions.

Example 2

[0187] The following example demonstrates that POMC null mice areprotected from the development of obesity-induced insulin resistance,and that the administration of melanocortin stimulating hormone (MSH) tothe mice nearly normalizes the glucoregulatory response in the mice.

[0188] Normal Glucose and Insulin Levels in POMC Null Mutants.

[0189] In the experiment illustrated in FIGS. 4A-4D, blood glucose andplasma insulin levels were measured in 5 months old females, 5 pergroup, either in the morning (fed state) or after an overnight fast(fasted). Mice were POMC null mutants (−/−), and heterozygous (+/−) andwildtype (+/+) littermates; as well as ob/ob mutants and wildtype orheterozygous (+/?) littermates as controls. Blood glucose was measuredusing Bayer Glucometer Elite; plasma insulin was determined using theLinco RIA kit. The results demonstrate the POMC null mutants have normalglucose and insulin levels as compared to their heterozygous andwildtype controls.

[0190] Normoglycemia in POMC Null Mutants Throughout Their Life Span

[0191] To see whether POMC null mutants are hyperglycemic at any pointin their life, their blood was tested for glucose levels at differentages (3, 5, and 7 months). The results are shown in FIG. 5. Bloodglucose levels in POMC null mutants were at no time out of the range ofnormal values.

[0192] Lack of Glucoregulatory Response in POMC Null Mutants DuringInsulin Tolerance Test

[0193] In this experiment, illustrated in FIG. 6, food was taken away inthe morning from 5 month old females, 5 per group. 6 hrs later, the micewere injected with insulin (Human insulin, 1 Unit/kg, i.p.). Blood wascollected at times indicated and glucose measured using a Glucometer.The results showed that blood glucose levels were not significantlydifferent between wildtype and heterozygous mice. However, glucoselevels were significantly lower in POMC mutants compared to wildtypelittermates after the first 60 minutes past insulin injection (80 min:P<0.05; 120 min: P<0.0001; 180 min: P<0.0001).

[0194] Corticosterone Supplementation Does Not Lead to Diabetes

[0195] In the experiment illustrated in FIG. 7, POMC null mutants andwildtype littermate females, 5 per group, received drinking watercontaining corticosterone (25 μg per mL) or nothing in addition for 3½months, starting at 3 months of age. Blood glucose levels weredetermined before and after corticosterone supplementation. The resultsshow that the supplementation with corticosterone does not inducediabetes in the animals, indicating that the reduced corticosterone inthe null mutant mice is not responsible for the lack ofinsulin-resistance or diabetes in the mice.

[0196] Corticosterone Supplementation Does Not Normalize theCounterregulatory Response

[0197] In the next experiment, illustrated in FIG. 8, when an insulintolerance test (ITT) was performed after corticosterone supplementation,it was determined that the blood glucose levels of both wildtypes andmutants do not fall to as low a level as in the untreated group, suchthat POMC null mutants do not die of hypoglycemia. However, the POMCmutants lag behind the wildtype mice in their counterregulatory responsein the same manner as without corticosterone supplementation.

[0198] MSH Almost Normalizes the Glucoregulatory Response

[0199] In this experiment, illustrated in FIG. 9, glucose was measuredin 4-6 month old female mice, wildtypes and POMC mutants, 5 mice pergroup. Mice were fasted for 5 hours. Each genotype (wildtype +/+ andmutant −/−) received either: (1) 0.1 mL saline i.p.; (2) 0.1 mL salinecontaining 1 μg α-MSH i.p.; or (3) 0.1 mL saline containing 1 μg ACTHs.c.

[0200] One hour later, human insulin (0.5 mIU/g body weight) wasinjected intraperitoneally in all mice, and glucose was measured at theindicated intervals in blood collected from a tail nick. The resultsshowed that blood glucose levels were not significantly differentbetween the MSH-reconstituted POMC mutants and their wildtypelittermates. Significant differences were found only between POMCwildtypes and either (1) saline- or (2) ACTH-treated mutants(saline-treated wildtype vs. mutant at 90 min: P<0.05, 120 min: P<0.01,180 min: P<0.05; ACTH-treated wildtype versus mutants at 90 min: P<0.01,120 min: P<0.05, 180 min: P<0.05).

[0201] In summary, in mice, as in humans, obesity is frequentlyaccompanied by insulin-resistant, type II diabetes. The presentinventors determined blood glucose levels and plasma insulin levels inPOMC null mutant mice, their wildtype and heterozygous littermates, andfor comparison, in ob/ob mutant mice and their littermates. Glucose andinsulin levels in both fed and fasting states were within normal rangesin pomc mutants; the ob/ob mutants showed the expected hyperglycemia andhyperinsulinemia (FIGS. 4A-4D).

[0202] POMC null mice have normal circulating levels of glucose andinsulin, in both the fed and fasted states. The results described abovedemonstrate that the genetic absence of POMC peptides can prevent thedevelopment of insulin resistance associated with obesity. Potentialbases for this protection are: 1) insulin production in the pancreas; 2)insulin signaling in insulin-sensitive tissues, such as muscle, adiposetissue, and liver; and 3) lipid metabolism in adipose tissue and liver.Restoration of MSH in the POMC mice by peripheral administration ofα-MSH restored the glucoregulatory ability of the mice, indicating thatthe biological activity of MSH contributes to insulin-resistance inobesity and diabetes.

[0203] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention.

1 8 1 5 PRT Artificial sequence DOMAIN (1)..(5) conserved region 1 GluHis Phe Arg Trp 1 5 2 13 PRT Homo sapiens 2 Ser Tyr Ser Met Glu His PheArg Trp Gly Lys Pro Val 1 5 10 3 7 PRT Artificial sequence MOD_RES(1)..(1) Xaa = Nle 3 Xaa Xaa His Xaa Arg Trp Xaa 1 5 4 13 PRT Artificialsequence VARIANT (4)..(4) Xaa = Met, Nle, or Cys 4 Ser Tyr Ser Xaa GluHis Phe Arg Trp Gly Lys Pro Val 1 5 10 5 7 PRT Artificial sequenceMOD_RES (1)..(1) Nle 5 Xaa Asp His Xaa Arg Trp Lys 1 5 6 7 PRTArtificial sequence MOD_RES (1)..(1) Xaa = Nle 6 Xaa Asp His Phe Arg TrpLys 1 5 7 845 DNA Mus musculus 7 gccaggcttg gctcactcgc ctggcctccctacaggcttg catccgggct tgcaaactcg 60 acctctcgct ggagacgccc gtgtttcctggcaacggaga tgaacagccc ctgactgaaa 120 acccccggaa gtacgtcatg ggtcacttccgctgggaccg cttcggcccc aggaacagca 180 gcagtgctgg cagcgcggcg cagaggcgtgcggaggaaga ggcggtgtgg ggagatggca 240 gtccagagcc gagtccacgc gagggcaagcgctcctactc catggagcac ttccgctggg 300 gcaagccggt gggcaagaaa cggcgcccggtgaaggtgta ccccaacgtt gctgagaacg 360 agtcggcgga ggcctttccc ctagagttcaagagggagct ggaaggcgag cggccattag 420 gcttggagca ggtcctggag tccgacgcggagaaggacga cgggccctac cgggtggagc 480 acttccgctg gagcaacccg cccaaggacaagcgttacgg tggcttcatg acctccgaga 540 agagccagac gcccctggtg acgctcttcaagaacgccat catcaagaac gcgcacaaga 600 agggccagtg agggtgcagg ggtcttctcattccaaggcc ccctccctgc atgggcgagc 660 tgatgacctc tagcctctta gagttacctgtgttaggaaa taaaaccttt cagatttcac 720 agtcggctct gatcttcaat aaaaactgcgtaaataaagt caaaacacaa ctgtccagtt 780 acactatcac gtgaccagat gctagaatgtaaagaaaaca tttctcaacc tccttgcccc 840 agcaa 845 8 235 PRT Mus musculus 8Met Pro Arg Phe Cys Tyr Ser Arg Ser Gly Ala Leu Leu Leu Ala Leu 1 5 1015 Leu Leu Gln Thr Ser Ile Asp Val Trp Ser Trp Cys Leu Glu Ser Ser 20 2530 Gln Cys Gln Asp Leu Thr Thr Glu Ser Asn Leu Leu Ala Cys Ile Arg 35 4045 Ala Cys Lys Leu Asp Leu Ser Leu Glu Thr Pro Val Phe Pro Gly Asn 50 5560 Gly Asp Glu Gln Pro Leu Thr Glu Asn Pro Arg Lys Tyr Val Met Gly 65 7075 80 His Phe Arg Trp Asp Arg Phe Gly Pro Arg Asn Ser Ser Ser Ala Gly 8590 95 Ser Ala Ala Gln Arg Arg Ala Glu Glu Glu Ala Val Trp Gly Asp Gly100 105 110 Ser Pro Glu Pro Ser Pro Arg Glu Gly Lys Arg Ser Tyr Ser MetGlu 115 120 125 His Phe Arg Trp Gly Lys Pro Val Gly Lys Lys Arg Arg ProVal Lys 130 135 140 Val Tyr Pro Asn Val Ala Glu Asn Glu Ser Ala Glu AlaPhe Pro Leu 145 150 155 160 Glu Phe Lys Arg Glu Leu Glu Gly Glu Arg ProLeu Gly Leu Glu Gln 165 170 175 Val Leu Glu Ser Asp Ala Glu Lys Asp AspGly Pro Tyr Arg Val Glu 180 185 190 His Phe Arg Trp Ser Asn Pro Pro LysAsp Lys Arg Tyr Gly Gly Phe 195 200 205 Met Thr Ser Glu Lys Ser Gln ThrPro Leu Val Thr Leu Phe Lys Asn 210 215 220 Ala Ile Ile Lys Asn Ala HisLys Lys Gly Gln 225 230 235

What is claimed is:
 1. A method to identify compounds useful inregulating insulin resistance in obesity and type II diabetes,comprising: a. administering a compound having melanocyte stimulatinghormone (MSH) biological activity to a genetically modified non-humananimal comprising a genetic modification within two alleles of its Pomclocus, wherein said genetic modification results in an absence ofproopiomelanocortin (Pomc) peptide activity in said animal, and whereinadministration of said compound having MSH activity induces insulinresistance in said animal; b. administering a compound to be evaluatedto said non-human animal model; and, c. selecting compounds from (b)that decrease the insulin resistance in said non-human animal ascompared to in the absence of said compound of (b).
 2. The method ofclaim 1, wherein said genetic modification is selected from the groupconsisting of a deletion, an insertion, a substitution and an inversionof nucleotides in said Pomc locus.
 3. The method of claim 1, whereinsaid genetic modification is a deletion of a nucleic acid sequencewithin two alleles of said Pomc locus, wherein said deletion results inan absence of expression of Pomc peptides by said animal.
 4. The methodof claim 1, wherein said genetic modification is a deletion of a nucleicacid sequence comprising exon 3 of Pomc or a portion of exon 3 of Pomcsufficient to prevent expression of Pomc peptides by two alleles of thePomc locus.
 5. The method of claim 1, wherein said animal is a mouse,and wherein said genetic modification is a deletion from said genome ofexon 3 of Pomc (SEQ ID NO:7).
 6. The method of claim 1, wherein saidcompound having MSH biological activity is selected from the groupconsisting of: MSH, a biologically active fragment of MSH, a homologueof MSH, a peptide mimetic of MSH, a non-peptide mimetic of MSH, and afusion protein comprising an MSH protein or fragment thereof.
 7. Themethod of claim 1, wherein said compound of (a) having MSH biologicalactivity is α-MSH.
 8. The method of claim 1, wherein said compound of(b) to be evaluated is an antagonist of MSH biological activity.
 9. Themethod of claim 1, wherein said compound of (b) to be evaluated isadministered prior to the step of administering said compound of (a)having MSH biological activity.
 10. A method to decrease insulinresistance in a mammal, comprising administering to said mammal that hasinsulin resistance a therapeutic composition comprising an antagonist ofmelanocortin stimulating hormone (MSH) biological activity, wherein saidantagonist decreases insulin resistance in said mammal.
 11. The methodof claim 10, wherein said antagonist of melanocortin stimulating hormone(MSH) is selected from the group consisting of a fragment of MSH havingMSH antagonist action, a homologue of MSH having MSH antagonist action,a peptide mimetic of MSH having MSH antagonist action, a non-peptidemimetic of MSH having MSH antagonist action, and a fusion proteincomprising any of said MSH antagonist compounds.
 12. The method of claim10, wherein said antagonist of MSH is a soluble MSH receptor or fragmentthereof that binds MSH.
 13. The method of claim 10, wherein saidantagonist of MSH is an antibody that selectively binds to MSH andthereby reduces or blocks the activity of MSH.
 14. The method of claim10, wherein said antagonist of MSH is an antibody that selectively bindsto a receptor for MSH and reduces or blocks the ability of MSH to bindto said receptor.
 15. The method of claim 10, wherein said therapeuticcomposition is administered transdermally.
 16. The method of claim 10,wherein said therapeutic composition is administered topically.
 17. Themethod of claim 10, wherein said therapeutic composition is administeredparenterally.
 18. The method of claim 10, wherein said therapeuticcomposition is administered in a controlled release formulation.
 19. Themethod of claim 10, wherein said antagonist of melanocortin stimulatinghormone biological activity is administered in a dose of from about 0.1μg to about 10 mg per kg body weight of said animal.
 20. A method totreat diabetes associated with insulin resistance in a mammal,comprising administering to said mammal that has insulin resistance anddiabetes a therapeutic composition comprising an antagonist ofmelanocortin stimulating hormone (MSH) biological activity, wherein saidantagonist decreases insulin resistance in said mammal.